Exogenous Fibrolytic Enzymes and Length of Storage Affect the Nutritive Value and Fermentation Profile of Maize Silage

Round 1

Reviewer 1 Report (Previous Reviewer 4)

 This manuscript showed the high value for the storage of maize silages. And the quality of paper is also improved after revision. Therefore, I suggested that it just need to be minor modified.

1.     The background of exogenous fibrolytic enzymes should be more descript in the introduction.

Author Response

The background of exogenous fibrolytic enzymes should be more descript in the introduction.

Response: Thank you for your comment. We added more information about the exogenous fibrolytic enzymes on lines 53-56 in the Introduction.

Reviewer 2 Report (Previous Reviewer 2)

Thanks for the revision , basically according to the comments from the first revision.

Unfortunately, the correction equation for the corrected dry matter content according to Weißbach and Strubelt is not correct. There are two equations according to Weißbach, one for grass silage and one for corn silage. In this paper, the equation for grass silage was used. Why? Is there a plausible reason for this, with the background that the Weißbach equations have been derived exactly at these silages?

Author Response

Unfortunately, the correction equation for the corrected dry matter content according to Weißbach and Strubelt is not correct. There are two equations according to Weißbach, one for grass silage and one for corn silage. In this paper, the equation for grass silage was used. Why? Is there a plausible reason for this, with the background that the Weißbach equations have been derived exactly at these silages?

Response: The equation is correct. The maize silage equation was used in this study. 2,3-butanediol and esters were added to the equation. We have replaced the reference with the following:

Weissbach, F.; Strubelt, C. Correcting the dry matter content of maize silages as a substrate for biogas production. Landtechnik-Net. 2008, 63 (2),82–83.

Reviewer 3 Report (New Reviewer)

Maize silage is the most used roughage in the diets of dairy cows in Brazil and worldwide. Improvements in the nutritive value of maize silage can result in a reduction in diet costs and in greater milk production. So this topic is very interesting and important. The authors can improve it before publish it. 

1.  Introduction section needs more information to improve it.

2. Too much tables, it would be better to use figures instead of tables.  

Author Response

1. Introduction section needs more information to improve it.

Response: Thank you for your comment, and we have added more information about the exogenous fibrolytic enzymes on lines 53-56 in the Introduction.

2. Too much tables, it would be better to use figures instead of tables.

Response: Thanks for the comments. The authors prefer presenting the results in Tables. The manuscript has only 3 Tables.

Reviewer 4 Report (New Reviewer)

Maize silage is the predominant forage used in the dairy cattle diet in the whole world. Therefore, improving fiber digestibility in maize silage is an important aspect that has been explored since this could improve farm profitability. This study has an original idea, producing new and complementary data to the present literature regarding the effect of exogenous fibrolytic enzymes over storage length.

In general, the authors conducted the introduction efficiently, providing an essential background from the literature regarding the topic explored and demonstrating the gap that was explored in the present study. Also, the methodologies were appropriate and well described. However, I have several comments and suggestions that are presented below.

Introduction

Line 70: “nutritive” instead of “nutirive”

Materials and Methods

Lines 81-83: I would recommend the authors provide the concentration of WSC in the plant before ensiling.

Lines 82-83: I would recommend spelling all the abbreviations used here since it was the first time that they were used in the manuscript.

Results

Lines 203-206: The authors stated that treatments E150 and E200 showed a greater NDFD than control. However, I believe the NDFD for E150 and E200 was also greater than E100 since the value is lower than the control (as presented in the table). Therefore, I would recommend that the authors include E100 in the sentence.

All the other part of the Results sections is well written.

Discussion

Lines 279-280: The authors mentioned that there was no hydrolysis of hemicellulose differently from Weinberg and Chen's study. However, I would recommend the authors re-writing this sentence since the hemicellulose was not evaluated in the present manuscript. They might use another term, such as NDF, instead of hemicellulose.

Lines 312-313: This sentence sounds confusing: “The NDF digestibility was also reduced in the study of Lynch et al. [27]”. When I read it, I understood that the NDF digestibility was reduced by ensiling time; however, Lynch et al. did not evaluate NDF digestibility statistically over time. Therefore, I would recommend that the authors re-write this sentence.

Lines 302-303: How did the authors observe this effect? If this means numerically, I would suggest the authors clarify it in the sentence. Also, if it was numerically, the same happened for the d30 and should be included here.

Lines 317-318: Was the maize plant used in the present manuscript a BMR hybrid or not? I would recommend adding that in this sentence to complement; therefore, the readers would better understand your results compared with the previous studies.

Lines 321-323: How about the difference between the methods? Der Bedrosian et al. carried out an in vitro digestibility assay, while the present manuscript evaluated the digestibility in situ. The authors might explore it in the justification as well.

Line 327: How do the authors explain that only the lower levels of EFE improved the starch digestibility vs. control at 60 d after ensiling, whereas there was no difference between the control and other EFE levels? Some studies in the literature also show a similar situation for other variables. I would recommend that the authors add a sentence explaining it or at least compare it with the other studies.

I agree that we should avoid discussing anything based on the numeric difference; however, the control treatment showed a greater NDF concentration than the other treatments that might come from hemicellulose or cellulose individually. As the EFE used in this study was a combination of hemicellulase and cellulase, I would like to know why the authors did not determine the ADF and lignin to estimate the hemicellulose and cellulose concentrations individually. I recommend that the authors add hemicellulose and cellulose concentrations to the study. I believe they would help in the discussion of several points.

Author Response

Introduction

Line 70: “nutritive” instead of “nutirive”

Response: Thank you for your suggestion, the word was corrected in the manuscript.

Materials and Methods

Lines 81-83: I would recommend the authors provide the concentration of WSC in the plant before ensiling.

Response: Unfortunately, WSC analysis of plant before ensiling was not performed.

Lines 82-83: I would recommend spelling all the abbreviations used here since it was the first time that they were used in the manuscript.

Response: Thank you for your recommendation. We have defined the abbreviations.

Results

Lines 203-206: The authors stated that treatments E150 and E200 showed a greater NDFD than control. However, I believe the NDFD for E150 and E200 was also greater than E100 since the value is lower than the control (as presented in the table). Therefore, I would recommend that the authors include E100 in the sentence.

Response: Thank you for your observation. We have included the E100 treatment in the sentence.  

All the other part of the Results sections is well written.

 Discussion

Lines 279-280: The authors mentioned that there was no hydrolysis of hemicellulose differently from Weinberg and Chen's study. However, I would recommend the authors re-writing this sentence since the hemicellulose was not evaluated in the present manuscript. They might use another term, such as NDF, instead of hemicellulose.

Response: As you suggested, the term hemicellulose was replaced by NDF.

Lines 312-313: This sentence sounds confusing: “The NDF digestibility was also reduced in the study of Lynch et al. [27]”. When I read it, I understood that the NDF digestibility was reduced by ensiling time; however, Lynch et al. did not evaluate NDF digestibility statistically over time. Therefore, I would recommend that the authors re-write this sentence.

Response: As you recommend, the sentence was rewritten.

Lines 302-303: How did the authors observe this effect? If this means numerically, I would suggest the authors clarify it in the sentence. Also, if it was numerically, the same happened for the d30 and should be included here.

Response: Since the interaction between dose and storage length was not statically significant, the authors decided to remove the sentence.

Lines 317-318: Was the maize plant used in the present manuscript a BMR hybrid or not? I would recommend adding that in this sentence to complement; therefore, the readers would better understand your results compared with the previous studies.

Response: The maize plant was not a BMR hybrid. The information was inserted in the sentence (Line 324).

Lines 321-323: How about the difference between the methods? Der Bedrosian et al. carried out an in vitro digestibility assay, while the present manuscript evaluated the digestibility in situ. The authors might explore it in the justification as well.

Response: We appreciate your comment and we have added information about the different methods between the studies on lines 328 - 330.

Line 327: How do the authors explain that only the lower levels of EFE improved the starch digestibility vs. control at 60 d after ensiling, whereas there was no difference between the control and other EFE levels? Some studies in the literature also show a similar situation for other variables. I would recommend that the authors add a sentence explaining it or at least compare it with the other studies.

Response: An explanation about this effect was inserted on lines 336 -340.

I agree that we should avoid discussing anything based on the numeric difference; however, the control treatment showed a greater NDF concentration than the other treatments that might come from hemicellulose or cellulose individually. As the EFE used in this study was a combination of hemicellulase and cellulase, I would like to know why the authors did not determine the ADF and lignin to estimate the hemicellulose and cellulose concentrations individually. I recommend that the authors add hemicellulose and cellulose concentrations to the study. I believe they would help in the discussion of several points.

Response: Thank you for your observation. Unfortunately, ADF and ADL analyses were not performed. Therefore, we could not calculate the concentrations of hemicellulose and cellulose.

Round 2

Reviewer 3 Report (New Reviewer)

I have no suggestions after the authors improved it. 

Reviewer 4 Report (New Reviewer)

The authors did a great job making modifications according to the suggestion from the previous revision. Also, they exceptionally addressed the questions that were pointed out. Therefore, I do not have any other suggestions.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

the manuscript about " Exogenous fibrolytic enzymes and length of storage affect the nutritive value and fermentation profile of maize silage" is well written and presented in a scientific way. the justification for doing such an experiment is understandable however, many studies were conducted to improve silage nutritive value. 

I have the following questions:

• did you consider that enzymes will reach their full capacity at some point and the length of ensiling will not be significant?
• Was 60-day period the best in the DM value due to enzymatic treatment? as mentioned by first point.
• I think to test different levels of enzymes could be more significant than the length of ensiling, also it can be shortened.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 2 Report

  The article shows a good experimental design, description of the chosen methods and results.

However, a critical evaluation of the fermentation quality characteristics presented in the paper is requested. Are the statistically confirmed results really biologically relevant?

Does the reduction of 0.08% ethanol in the control reported in the paper have any biological or practical significance? Are the values correctly analyzed and calculated?

Aren't the statistically confirmed differences in this lowest concentration range a result of very clean experimental conditions, with little scatter due to experimental design?

Are the conclusions transferable to ethanol and ester contents as they exist in the literature for practical silages (many times higher than the values in this paper)?

Abstract:

In the abstract as well as in the conclusions, statements are formulated without concentration data/concrete results. This generalization is not acceptable from my point of view.

Line 15: What is the main reason for adding fibrolytic enzymes to corn? The improvement of the fermentation quality or the improvement of the feed value? In my opinion, the improvement of the feed value should be in the foreground, everything else makes no sense with corn.

Introduction:

Line 46/47: The need for higher concentrations of water-soluble carbohydrates in corn is to be discussed. Normally, the concentrations of fermentable substrate in corn silage are sufficient. On the contrary, there is a risk that corn silages with a lot of water-soluble carbohydrates are all the more exposed to aerobic spoilage. Yeasts also use lactic acid as a substrate.

Material and methods:

Line 97: Can you please report whether NDF was performed with or without ashing at the end of the analysis?

Line 107- 117: Please indicate the limits of quantitation/detection for each of the methods listed here.

This is all the more important as relatively very low values of e.g. alcohols and esters are postulated.

Line 117-119: Please check the equation and the reference concerned (Weißbach); in the Weißbach equation, for example, neither esters nor 2.3. Butandiol are included

Results:

Line 217- 231: as already explained above, the relevance of the analyzed contents/concentration changes described is questioned, especially for ethanol

Discussion:

Line 238-249: Against the background of the statistically significant but questionable biologically relevant changes in the characteristics of feed value/digestibility in this paper, it is desirable to show the discussed changes in the cited paper in more detail (concentration data). In which range did the authors find effects of storage time?

Line 260-265: To what extent was the NDF content reduced in the studies cited? Was the water-soluble carbohydrate content also reported?

Line 287-290: Protein degradation during ensiling is a current topic, current papers could still be included here.

Line 293-295: please check this statement (ph highest on day 90?) lactic acid lowest...see table 2

Line 301-306: here orders of magnitude (concentration data in comparison would be very interesting)

Line 327-333: the formation of ethanol can also occur by heterofermentative lactic acid bacteria; the detected contents of 1,2-propanediol indicate the occurrence of heterofermentative LAB, especially since no silage additive with heterofermenters was added ; it was also son discussed by you in line 307-314; whether a yeast reduction took place here is purely speculative and should also not be discussed prior in view of the low ethanol contents

Line 342-344: There are further papers from this working group (Weiß et. al.) and also Hafner et. al. which could be used to classify the present concentration ratios.

Line 346-347: please check: is the increase in butyric acid significant from 60-90 days (all letters must be different); however, regardless, these are biologically irrelevant magnitudes

Conclusion:

Is the acetic acid concentration really increased significantly? This is also not clear in the discussion.

again, statements are formulated without concentration data/concrete results. This generalization is not acceptable from my point of view.

Author Response

Point 1: The article shows a good experimental design, description of the chosen methods and results.

However, a critical evaluation of the fermentation quality characteristics presented in the paper is requested. Are the statistically confirmed results really biologically relevant?

Does the reduction of 0.08% ethanol in the control reported in the paper have any biological or practical significance? Are the values correctly analyzed and calculated?

Aren't the statistically confirmed differences in this lowest concentration range a result of very clean experimental conditions, with little scatter due to experimental design?

Are the conclusions transferable to ethanol and ester contents as they exist in the literature for practical silages (many times higher than the values in this paper)?

Response 1: The results were discussed considering the statistical differences, which represent the biological trends induced by the treatments. We agree that differences were not large in some cases. Hence, we included a sentence to warn the readers.

Abstract:

Point 2:  In the abstract as well as in the conclusions, statements are formulated without concentration data/concrete results. This generalization is not acceptable from my point of view.

Response 2: The lack of concentration data and concrete results in the abstract is due to the restriction of 200 words.

Point 3:  Line 15: What is the main reason for adding fibrolytic enzymes to corn? The improvement of the fermentation quality or the improvement of the feed value? In my opinion, the improvement of the feed value should be in the foreground, everything else makes no sense with corn.

Response 3: The idea of adding xylanase and ß-glucanase to corn silage is based on the improvement of both feed value and fermentation products. The improvement of feed value is paramount from the animal nutrition point of view. However, changes in fermentative profile, which could increase aerobic stability in corn silages, are also important.

Introduction:

Point 4:  Line 46/47: The need for higher concentrations of water-soluble carbohydrates in corn is to be discussed. Normally, the concentrations of fermentable substrate in corn silage are sufficient. On the contrary, there is a risk that corn silages with a lot of water-soluble carbohydrates are all the more exposed to aerobic spoilage. Yeasts also use lactic acid as a substrate.

Response 4: As suggested, the sentence was removed.

Material and methods:

Point 5: Line 97: Can you please report whether NDF was performed with or without ashing at the end of the analysis?

Response 5: The information was included in the manuscript. It was performed without ashing.

Point 6: Line 107- 117: Please indicate the limits of quantitation/detection for each of the methods listed here. This is all the more important as relatively very low values of e.g. alcohols and esters are postulated.

Response 6: As suggested the information was included in the manuscript. The detection limit of trace compounds (e.g. esters) was 1 mg/kg DM.

Point 7: Line 117-119: Please check the equation and the reference concerned (Weißbach); in the Weißbach equation, for example, neither esters nor 2.3. Butandiol are included

Response 7: All compounds listed in the equation were considered by Weissbach, except esters. We adapted the equation also considering esters, as they are volatile during oven drying. The sentence was rephrased in the manuscript.

Results:

Point 8: Line 217- 231: as already explained above, the relevance of the analyzed contents/concentration changes described is questioned, especially for ethanol

Response 8: The results were discussed considering the statistical differences, which represent the biological trends induced by the treatments. We agree that differences were not large in some cases. Hence, we included a sentence to warn the readers.

Discussion:

Point 9: Line 238-249: Against the background of the statistically significant but questionable biologically relevant changes in the characteristics of feed value/digestibility in this paper, it is desirable to show the discussed changes in the cited paper in more detail (concentration data). In which range did the authors find effects of storage time?

Response 9: We appreciate your suggestions, and the information will be added to the manuscript.

Point 10: Line 260-265: To what extent was the NDF content reduced in the studies cited? Was the water-soluble carbohydrate content also reported?

Response 10: The NDF content reduction in these studies varied from 3% to 20% in comparison to the control treatment. Water-soluble carbohydrates increase were reported and ranged from 9% to 393% in comparison to the control.

Point 11: Line 287-290: Protein degradation during ensiling is a current topic, current papers could still be included here.

Response 11: We appreciate your suggestions, and we will find more time to research in the literature.

Point 12: Line 293-295: please check this statement (ph highest on day 90?) lactic acid lowest...see table 2

Response 12: The statement was checked and complemented in the manuscript.

Point 13: Line 301-306: here orders of magnitude (concentration data in comparison would be very interesting)

Response 13: The statement was complemented in the manuscript.

Point 14: Line 327-333: the formation of ethanol can also occur by heterofermentative lactic acid bacteria; the detected contents of 1,2-propanediol indicate the occurrence of heterofermentative LAB, especially since no silage additive with heterofermenters was added ; it was also son discussed by you in line 307-314; whether a yeast reduction took place here is purely speculative and should also not be discussed prior in view of the low ethanol contents

Response 14: As suggested, the sentence was removed.

Point 15: Line 342-344: There are further papers from this working group (Weiß et. al.) and also Hafner et. al. which could be used to classify the present concentration ratios.

Response 15: We appreciate your suggestions, and information on both papers will be added to the manuscript.

Point 16: Line 346-347: please check: is the increase in butyric acid significant from 60-90 days (all letters must be different); however, regardless, these are biologically irrelevant magnitudes

Response 16: The statement was checked and corrected in the manuscript.

Conclusion:

Point 17: Is the acetic acid concentration really increased significantly? This is also not clear in the discussion.

Response 17: The results were discussed considering the statistical differences, which represent the biological trends induced by the treatments. We agree that differences were not large in some cases. Hence, we included a sentence to warn the readers.

Point 18: again, statements are formulated without concentration data/concrete results. This generalization is not acceptable from my point of view.

Response 18: The corrections were made in the manuscript as suggested.

Reviewer 3 Report

In this manuscript, the effects of exogenous fibrolytic enzymes (EFE) and length of storage on the quality of maize silage was investigated and the fermentative profile and the nutritive value were tested. This was a general study and no highlights were provided. However, if the authors can provide more information or data, the readers may find more interesting and useful information from this study. So, this MS cannot be accepted at this stage. The authors need pay attention to the following points.

1. Line 20, Page 1: In Abstract, the abbreviation “DM” should not be used for its first appearance.
2. Line 21, Page 1: this sentence was not reasonable, please check it. “Length of storage” should be revised and can be replaced by “increased/prolonged length of storage”.
3. Lin 69-73, Page 2: what is the purpose of the application of exogenous fibrolytic enzymes? It is obvious that only EFE cannot increase the DM loss due to the presence of lignin in maize silage because lignin prevented the hydrolysis of cellulose and hemicellulose. And also, it was proved by the results and described in the conclusion- “The EFE application in maize silage did not increase the digestibility of nutrients”. Therefore, the selection of enzyme was reasonable or not?
4. Page 5: in Section 3.2, no microbial analysis was conducted, which is important for the illustration to the results of fermentative profile. It was suggested to be added.
5. In Section 4:

1) Is it economic to add the EFE to the maize silage? It should be discussed and analyzed.

2) As described, The EFE application in maize silage increased the acetic acid content and decreased the contents of ethanol, ethyl lactate and ethyl acetate. Is acetic acid beneficial for the maize silage? Or can it increase the nutritional value to the maize silage? It should be discussed in details. 

Author Response

Point 1:  Line 20, Page 1: In Abstract, the abbreviation “DM” should not be used for its first appearance.

Response 1: As suggested the correction was made in the manuscript.

Point 2:  Line 21, Page 1: this sentence was not reasonable, please check it. “Length of storage” should be revised and can be replaced by “increased/prolonged length of storage”.

Response 2: As suggested the correction was made in the manuscript.

Point 3:  Lin 69-73, Page 2: what is the purpose of the application of exogenous fibrolytic enzymes? It is obvious that only EFE cannot increase the DM loss due to the presence of lignin in maize silage because lignin prevented the hydrolysis of cellulose and hemicellulose. And also, it was proved by the results and described in the conclusion- “The EFE application in maize silage did not increase the digestibility of nutrients”. Therefore, the selection of enzyme was reasonable or not?

Response 3: Although DM losses did not occur by applying EFE to corn silage, the increase in acetic acid by the enzymes, could lead to an increase in DM losses, since carbon dioxide is released in the fermentation process. The idea of applying EFE to corn silages was to evaluate the solubilization of carbohydrates and the consequences of it the fermentative profile.

Point 4:  Page 5: in Section 3.2, no microbial analysis was conducted, which is important for the illustration to the results of fermentative profile. It was suggested to be added.

Response 4: The sentence was removed since there was no microbial analysis.

Point 5:  In Section 4:

1) Is it economic to add the EFE to the maize silage? It should be discussed and analyzed.

2) As described, The EFE application in maize silage increased the acetic acid content and decreased the contents of ethanol, ethyl lactate and ethyl acetate. Is acetic acid beneficial for the maize silage? Or can it increase the nutritional value to the maize silage? It should be discussed in details. I found that sections 2 & 3 should be re‐organized and be shortened. It may be easier for the readers if the authors define properly the mixture of regression model and the class‐ membership equation first before moving to the computation of the GINI and of the Polarization of subgroups. Sections 2.1 and 2.2 are too long and can be significantly reduced. In section 2.1 the authors assume the condition u_k > u_j, but this does not appear anywhere else in the calculation of the mixture of regression model. After equation (10) all the other equations are not numbered.

Response 1: The economic evaluation of EFE inclusion was not performed, because the benefit of the increase in acetic acid in corn silages would be shown in aerobic stability, which was not evaluated. The other suggestions of re-organizing and shortening sections 2 and 3 will be evaluated.

Reviewer 4 Report

This article deals with the effect of maize silage storage with exogenous fibrolytic enzymes. The scientific novelty of the manuscript is lower, but it provides high industrial value. Therefore, the manuscript needs to be minor modified.

1. In table 1-3, please explain why is there no standard deviation in the data?
2. I would like to suggest to the authors consider conversing some information in the table to figure. Table is difficult to see the trend of data.
3. I would like to suggest that can use some other methodology in your future work such as response surface methodology or Taguchi method to evaluate the interaction of factors and its optimization.

Author Response

Point 1: In table 1-3, please explain why is there no standard deviation in the data?

Response 1: To measure the statistical dispersion we used the standard error of means (SEM), which is included in each table.

Point 2: I would like to suggest to the authors consider conversing some information in the table to figure. Table is difficult to see the trend of data.

Response 2: At first, figures were used to describe some variables. However, the authors decided to standardize all the data in tables, to shorten the manuscript, otherwise would be necessary more figures than tables.

Point 3: I would like to suggest that can use some other methodology in your future work such as response surface methodology or Taguchi method to evaluate the interaction of factors and its optimization.

Response 3: We appreciate your suggestions

Round 2

Reviewer 2 Report

The indications from the review process were only partially and very marginally implemented.
The generalization of the results in the abstract, line 21-27, is not acceptable. All recommendations/comments for critical discussion of the silage quality characteristics evaluated in this paper, in particular the volatile organic compounds ethanol, ethyl ester and acetic acid, have been ignored. No references to commonly occurring levels of VOCs were made/discussed. The conclusions in the present form lead to the fundamental question of the value of the present work. 
The conclusions were changed and supported with concrete results, but this is not reflected in the abstract. The results in the abstract, which are only general (without concentration data on ethanol, ester and acetic acid content), convey to the reader that a reduction of these VOCs is possible by enzyme addition. Due to the practically irrelevant orders of magnitude, the reviewer strongly doubts whether this statement is generally valid.

Author Response

Point 1: The indications from the review process were only partially and very marginally implemented.

The generalization of the results in the abstract, line 21-27, is not acceptable. All recommendations/comments for critical discussion of the silage quality characteristics evaluated in this paper, in particular the volatile organic compounds ethanol, ethyl ester and acetic acid, have been ignored. No references to commonly occurring levels of VOCs were made/discussed. The conclusions in the present form lead to the fundamental question of the value of the present work.

The conclusions were changed and supported with concrete results, but this is not reflected in the abstract. The results in the abstract, which are only general (without concentration data on ethanol, ester and acetic acid content), convey to the reader that a reduction of these VOCs is possible by enzyme addition. Due to the practically irrelevant orders of magnitude, the reviewer strongly doubts whether this statement is generally valid.

Response 1: Thank you for your further comments, which have helped us to improve our manuscript. We have revised the manuscript accordingly. The abstract was implemented with the results as suggested, to avoid generalization (line 21 -33). Also, a paragraph was included in the discussion section about the VOCs levels found in the literature suggested ( line 382 - 389).  

Reviewer 3 Report

The authors provided response to some of the reviewers’ comments. However, no great improvement was conducted, the readers cannot find interesting and useful information from this study due to the limited data and information. So, this MS cannot be accepted at this stage.

1. Since the idea of applying EFE to corn silages was to evaluate the solubilization of carbohydrates and the consequences of it the fermentative profile, the microbial analysis should be conducted to evaluate the effect of applying EFE on its fermentation process and explain the reason for the difference of metabolites. At least, more literatures should be cited and more discussion should be provided. However, the author didn’t provide the data and only deleted the related sentences.
2. As described by the authors, the benefit of the increase in acetic acid in corn silages would be shown in aerobic stability due to the applying EFE. In fact, applying sodium acetate compounds can also improve the quality and aerobic stability of silage. So, it is important to do the economic analysis. At least, more discussion should be provided.
3.  The authors made something wrong with the Point 5, some comments were not belonging to this reviewer. 

Author Response

The authors provided response to some of the reviewers’ comments. However, no great improvement was conducted, the readers cannot find interesting and useful information from this study due to the limited data and information. So, this MS cannot be accepted at this stage.

Response: Tha authors acknowledge the reviewer´s comments, which have helped us to improve our manuscript. Please, see the author respnses below.

Point 1:  Since the idea of applying EFE to corn silages was to evaluate the solubilization of carbohydrates and the consequences of it the fermentative profile, the microbial analysis should be conducted to evaluate the effect of applying EFE on its fermentation process and explain the reason for the difference of metabolites. At least, more literatures should be cited and more discussion should be provided. However, the author didn’t provide the data and only deleted the related sentences.

Response 1: The authors agree that microbial analyses could explain some effects in the corn silages treated with EFE, mainly related to the reduction of ethanol. Although microbial analyses have not been conducted, we still believe that the effect of EFE on the fermentation profile gives relevant results that cen be used by the scientific community. More references have been added, for example regarding esters, to improve the discussion.

Point 2:  As described by the authors, the benefit of the increase in acetic acid in corn silages would be shown in aerobic stability due to the applying EFE. In fact, applying sodium acetate compounds can also improve the quality and aerobic stability of silage. So, it is important to do the economic analysis. At least, more discussion should be provided.

Response 2: The authors appreciate your suggestion. However, aerobic stability was not performed due to the small amount of silage in each bag (approximately 500g). However, we added the following text with ciations on lines 349-350: Similar to storage length, the EFE application also increased the acetic acid content, which have been shown to increase aerobic stability of maize silage (Danner et al. 2002) because of its antifungal capacity (Krooneman et al. 2002).  If we consider corn silage price of R$ 370 per ton of DM, the breakeven point to pay the doses, 100, 150, 200g/ton DM, by reducing the DM losses in the feed-out phase would be, 3,5%, 5,3%, 7,0%, respectively. Also, we considered evaluating the economics using the DM losses data, during fermentation. It was not possible, because there was no effect of EFE on DM losses. We have chosen not to include economics in the manuscript as this was not part of the aim.

The authors made something wrong with the Point 5, some comments were not belonging to this reviewer. 

Response: We are sorry for this.