Dry Matter Content and Additives with Different Modes of Action Modify the Preservation Characteristics of Grass Silage

Round 1

Reviewer 1 Report

Introduction - introduction needs to be supplemented. The introduction does not provide an understanding of the problem the authors pose and decide in the study.

The literature citations start with #6, references 1, 3, 5 are not cited in text.

There is no reasoning for the use of resin acids. It is not clear why the authors included them in their study at all.

2. Materials and Methods - The article lacks a description of how the bacteria were introduced, was it a suspension, a lyophilic preparation? if it was a suspension, then in materials and methods you need to insert a section like "suspension preparation" or culturing bacteria for inolulation.

LAB analysis methodology is not described, while the author writes that the number of bacteria at the beginning of the experiment was in the minimum of 2 × 105 cfu/g of fresh forage.

I think that the methods Lactic acid was analyzed according to Haacker et al. [17] and ammonia according to McCullough [18] should be presented in more detail, because the references are controversial (Haacker - publication in Finnish; McCullough - method for blood)

There is no clear rationale for the use of experiment 4 and 5, as marco et al., 2022 [34] clearly show the role of these additives on silage from these grasses.(4. Salt-based additive (SA; sodium benzoate, potassium sorbate and sodium nitrite; Safesil Pro, Salinity AB, Göteborg, Sweden at 5 l/t) 5. Formic and propionic acid-based additive (FPA; formic acid, propionic acid, sodium formate and potassium sorbate; AIV Ä ssä Na, Eastman, Oulu, Finland at 5 l/t)).

Fig.1 and 2 - it is not very clear how the Standard error of the mean can be the same for all variants?

P395-400- the citation was not used correctly, there is no mention of red clover silages in these articles.

3.2.2. Experiment 2 – Resin Acids Used as Silage Additives - The presence of this part of the study is very controversial.

In conclusion, the article is very poorly developed for the level of the journal Fermentation, previously published results are repeated, and there is not enough discussion of the results.

Author Response

Reviewer #1:

Introduction - introduction needs to be supplemented. The introduction does not provide an understanding of the problem the authors pose and decide in the study.

Reply: Thank you for your comment. An entire paragraph emphasizing the importance of studies in silage making has been added to the Introduction, and in the end, the objectives were clarified.

 

The literature citations start with #6, references 1, 3, 5 are not cited in text.

Reply: You are right. It was my mistake forgetting to update Mendeley once more before the submission. My apologies and thanks for highlighting this issue. It is now corrected.

 

There is no reasoning for the use of resin acids. It is not clear why the authors included them in their study at all.

Reply: We need to develop silage additives with different principles, which would control or modulate the fermentation pattern of the silages. We speculated that resin acids are one possibility, however they have not been tested as silages additives. Then, in order to express the importance of resin acids in this study, we added to the Introduction the following quote: “Resin acids are defence chemicals of coniferous trees, and it has been hypothesized that they could positively affect animal production, although effects on ruminants have not been proven. Because of their antimicrobial activity, resin acids may have potential to improve silage preservation quality and aerobic stability.”

 

2. Materials and Methods - The article lacks a description of how the bacteria were introduced, was it a suspension, a lyophilic preparation? if it was a suspension, then in materials and methods you need to insert a section like "suspension preparation" or culturing bacteria for inolulation.

Reply: Thanks for the comment, it gives us a chance to better clarify it. Those are commercial products and according to manufactures’ instructions, they are simply diluted in water and sprayed in the grass material. This statement is included in the text.

 

LAB analysis methodology is not described, while the author writes that the number of bacteria at the beginning of the experiment was in the minimum of 2 × 105 cfu/g of fresh forage.

Reply: Indeed, you are right, LAB analyses were not performed, because it is a given concentration provided in the commercial product indicated by the manufacturer.

 

I think that the methods Lactic acid was analyzed according to Haacker et al. [17] and ammonia according to McCullough [18] should be presented in more detail, because the references are controversial (Haacker - publication in Finnish; McCullough - method for blood)

Reply: Additional information was included in the text regarding these analyses.

 

There is no clear rationale for the use of experiment 4 and 5, as marco et al., 2022 [34] clearly show the role of these additives on silage from these grasses.(4. Salt-based additive (SA; sodium benzoate, potassium sorbate and sodium nitrite; Safesil Pro, Salinity AB, Göteborg, Sweden at 5 l/t) 5. Formic and propionic acid-based additive (FPA; formic acid, propionic acid, sodium formate and potassium sorbate; AIV Ä ssä Na, Eastman, Oulu, Finland at 5 l/t)).

Reply: We kindly do not fully agree with this statement, and gently express our opinion that it does not necessarily mean that results found once are conclusive results. We include similar treatments in order to use them as a reference and also confirm that the same results are obtained in different situations with different types of plant material and potentially clearly deviating e.g. in the epiphytic microflora.

 

Fig.1 and 2 - it is not very clear how the Standard error of the mean can be the same for all variants?

Reply: This is an output of the MIXED procedure in SAS. We obtained the LS means for each treatment (Estimate) and then the same standard error for all the means, please see an example of SAS output bellow:

Solution for Fixed Effects
Effect	Treat	Estimate	Standard Error	DF	t Value	Pr > |t|	Alpha	Lower	Upper
Treat	1	3.9537	0.02197	17.4	179.93	<.0001	0.05	3.9074	3.9999
Treat	2	3.9337	0.02197	17.4	179.02	<.0001	0.05	3.8874	3.9799
Treat	3	3.9903	0.02197	17.4	181.6	<.0001	0.05	3.9441	4.0366
Treat	4	3.8237	0.02197	17.4	174.02	<.0001	0.05	3.7774	3.8699
Treat	5	3.8037	0.02197	17.4	173.11	<.0001	0.05	3.7574	3.8499
Treat	6	4.2937	0.02197	17.4	195.41	<.0001	0.05	4.2474	4.3399
Treat	7	4.597	0.02197	17.4	209.21	<.0001	0.05	4.5507	4.6433
Treat	8	4.297	0.02197	17.4	195.56	<.0001	0.05	4.2507	4.3433
Treat	9	4.1503	0.02197	17.4	188.88	<.0001	0.05	4.1041	4.1966
Treat	10	4.237	0.02197	17.4	192.83	<.0001	0.05	4.1907	4.2833

 

P395-400- the citation was not used correctly, there is no mention of red clover silages in these articles.

Reply: Authors’ ideas here was simply to show the effect of different additives on the extension of aerobic stability regardless of forage material utilized. Thus, we emphasized the effect of salt and formic acid on aerobic stability of both timothy grasses and red clover, as stated in the text.

 

3.2.2. Experiment 2 – Resin Acids Used as Silage Additives - The presence of this part of the study is very controversial.

Reply: We appreciate your comment, and we take a chance to explain that we included this dataset regarding resin acids to our manuscript in order to bring novelty as well as to state the need to develop additives with different modes of action. Hence, we truly believe that the Experiment 2 is very valid with the sense of innovation in silage making.

 

In conclusion, the article is very poorly developed for the level of the journal Fermentation, previously published results are repeated, and there is not enough discussion of the results.

Reply: With all due respect, we do not fully agree with the reviewer's view, but we respect his/her opinion. We believe that the manuscript is very clear, well written, with several important information in the different sections, and with results never described in the literature (such as the use of resin acids). Additionally, we believe that there is no lack of information, including the fact that the manuscript is already much longer than the average of articles published by Fermentation Journal (our manuscript is now up to 17 pages). We cordially express our compliments to the reviewer for his/her critical view of our work, and we also take the opportunity to say that this study required great financial and intellectual efforts, and that now, with great quality results it deserves to be published.

 

Final reply: Authors greatly appreciate comments and feedback from Reviewer #1 in improving our manuscript. Many thanks!

The authors.

Reviewer 2 Report

General comments: This article evaluates the effects of different management options on silage quality through two experiments, including dry matter (DM) content, types of additives, and the impact of resin acid products on silage characteristics and aerobic stability. The article is forward-looking, however, there are some areas that require modification.

 

Introduction

1、L25 There is insufficient information about the influence of DM content on microbial activity, fermentation efficiency, and storage stability in the silage process. It is recommended to provide additional details in this regard.

 

2、L29 More background information can be provided regarding the potential impact of climate change on agricultural production and silage management.

 

3、L39 More information can be provided about the characteristics of resin acids, their role in the silage process, and existing research in this area.

 

Results and Discussion

1、L187-189 Emphasize the significance and winter hardiness of this grass as a perennial forage in cool and humid regions.

 

2、L209-212 Highlight the importance of low counts of molds and yeasts in the raw material during the harvesting period, as silage feed may be exposed to aerobic conditions during practical use.

 

3、L269-274 It is advisable to further explain the biological mechanisms behind the decrease in ammonia nitrogen concentration due to the effects of FPA, such as the impact of acidification on reducing protein breakdown.

 

4、L276-279 Explain why even though ammonia nitrogen concentrations in C, HO, HE, and SA are higher than in FPA, they are still well-preserved.

 

5、L280-285 Further discuss the lack of signs indicating the activity of ammonia-producing bacteria in the current material.

Author Response

Reviewer #2:

General comments: This article evaluates the effects of different management options on silage quality through two experiments, including dry matter (DM) content, types of additives, and the impact of resin acid products on silage characteristics and aerobic stability. The article is forward-looking, however, there are some areas that require modification.

Reply: Dear reviewer, thank you for your constructive feedback. It’s very inspiring for the authors to have such a positive comment on our manuscript that required so many efforts. We have worked to attend all your requirements. Many thanks!

 

Introduction

1、L25 There is insufficient information about the influence of DM content on microbial activity, fermentation efficiency, and storage stability in the silage process. It is recommended to provide additional details in this regard.

Reply: Thanks for the valid comment. Additional information was included in the Introduction.

 

2、L29 More background information can be provided regarding the potential impact of climate change on agricultural production and silage management.

Reply: The second paragraph of Introduction emphasizes the challenges and potential impact of climate change on the silage production, which expresses the importance of different approaches in silage making research, such as forage dry matter content and innovative additives (factors in our experimental design).

 

3、L39 More information can be provided about the characteristics of resin acids, their role in the silage process, and existing research in this area.

Reply: Thank you so much for such a valid and useful comment. We need to develop silage additives with different principles, which would control or modulate the fermentation pattern of the silages. We speculated that resin acids are one possibility, however they have not been tested as silages additives yet, and that is why we could not cite existing research in this area. Then, in order to express the importance of resin acids in this study, we added to the Introduction the following quote: “Resin acids are defence chemicals of coniferous trees, and it has been hypothesized that they could positively affect animal production, although effects on ruminants have not been proven. Because of their antimicrobial activity, resin acids may have potential to improve silage preservation quality and aerobic stability.”

 

Results and Discussion

1、L187-189 Emphasize the significance and winter hardiness of this grass as a perennial forage in cool and humid regions.

Reply: The following sentence regarding timothy characteristics is in Results and Discussion: “Timothy grass is the most important perennial forage grown for silage and hay production in cool humid regions of Northern Europe, Asia and North America. The popularity of timothy in these areas is mainly because of its winter hardiness.”

 

2、L209-212 Highlight the importance of low counts of molds and yeasts in the raw material during the harvesting period, as silage feed may be exposed to aerobic conditions during practical use.

Reply: The fourth paragraph of Results and Discussion section is specifically about the microbial quality of the raw material, including the reviewer’s request: “Low counts of moulds and yeasts in the raw material during harvest facilitate adequate aerobic stability of the silage when opened and exposed to aerobic conditions during the feeding period.”

 

3、L269-274 It is advisable to further explain the biological mechanisms behind the decrease in ammonia nitrogen concentration due to the effects of FPA, such as the impact of acidification on reducing protein breakdown.

Reply: Thanks for the comment. The biological mechanism of this process is clearly stated in the text. Please see text highlighted in yellow.

 

4、L276-279 Explain why even though ammonia nitrogen concentrations in C, HO, HE, and SA are higher than in FPA, they are still well-preserved.

Reply: Basically because the difference between those treatments is rather small, additionally according to Wilkinson (1990), grass silage with ammonia-N in the range of 50 – 100 g/kg total N are regarded as well fermented. This statement indicates that even if ammonia-N in C, HO, HE and SA was higher than in FPA, they were still well preserved. This information is clearly presented and highlighted in yellow in the text.

 

5、L280-285 Further discuss the lack of signs indicating the activity of ammonia-producing bacteria in the current material.

Reply: We stated in the text that even Control silages did not indicate the presence of clostridial fermentation, which would in turn produce ammonia. Thus, this was an intrinsic characteristic of the raw material utilized in this study. Thanks for the comment and this information is now highlighted in yellow in the text.

 

Final reply: Authors greatly appreciate comments and feedback from Reviewer #2 in improving our manuscript. Many thanks!

The authors.

Round 2

Reviewer 1 Report

Given the authors' efforts to improve the article, corrections can be recommended to the journal. From myself, I recommend that in the future authors should build up the experimental material for articles in a more logical way.  There is no need to mix two different experiments, but if you have done so, the argumentation should be stronger than in the above paper. There should be logic and correlation in the paper, not just opportunities to extend any study. 

Author Response

Given the authors' efforts to improve the article, corrections can be recommended to the journal. From myself, I recommend that in the future authors should build up the experimental material for articles in a more logical way.  There is no need to mix two different experiments, but if you have done so, the argumentation should be stronger than in the above paper. There should be logic and correlation in the paper, not just opportunities to extend any study.

Reply: Dear Reviewer, thank you so much for your positive feedback and we fully agree with your advice. These considerations will be taken in our next experiments.

Kind regards,

The authors.

Reviewer 2 Report

General comments: This article evaluates the effects of different management options on silage quality through two experiments, including dry matter (DM) content, types of additives, and the impact of resin acid products on silage characteristics and aerobic stability. The article is forward-looking, however, there are some areas that require modification.

 

Introduction

1、L25 There is insufficient information about the influence of DM content on microbial activity, fermentation efficiency, and storage stability in the silage process. It is recommended to provide additional details in this regard.

 

2、L29 More background information can be provided regarding the potential impact of climate change on agricultural production and silage management.

 

3、L39 More information can be provided about the characteristics of resin acids, their role in the silage process, and existing research in this area.

 

Results and Discussion

1、L187-189 Emphasize the significance and winter hardiness of this grass as a perennial forage in cool and humid regions.

 

2、L209-212 Highlight the importance of low counts of molds and yeasts in the raw material during the harvesting period, as silage feed may be exposed to aerobic conditions during practical use.

 

3、L269-274 It is advisable to further explain the biological mechanisms behind the decrease in ammonia nitrogen concentration due to the effects of FPA, such as the impact of acidification on reducing protein breakdown.

 

4、L276-279 Explain why even though ammonia nitrogen concentrations in C, HO, HE, and SA are higher than in FPA, they are still well-preserved.

 

5、L280-285 Further discuss the lack of signs indicating the activity of ammonia-producing bacteria in the current material.

• Please double check to make the article more fluent.

Author Response

General comments: This article evaluates the effects of different management options on silage quality through two experiments, including dry matter (DM) content, types of additives, and the impact of resin acid products on silage characteristics and aerobic stability. The article is forward-looking, however, there are some areas that require modification.

Reply: Dear reviewer, thank you for your constructive feedback. It’s very inspiring for the authors to have such a positive comment on our manuscript that required so many efforts. We have worked to attend all your requirements. Many thanks!

 

Introduction

1、L25 There is insufficient information about the influence of DM content on microbial activity, fermentation efficiency, and storage stability in the silage process. It is recommended to provide additional details in this regard.

Reply: Thanks for the valid comment. Additional information was included in the Introduction.

 

2、L29 More background information can be provided regarding the potential impact of climate change on agricultural production and silage management.

Reply: The second paragraph of Introduction emphasizes the challenges and potential impact of climate change on the silage production, which expresses the importance of different approaches in silage making research, such as forage dry matter content and innovative additives (factors in our experimental design).

 

3、L39 More information can be provided about the characteristics of resin acids, their role in the silage process, and existing research in this area.

Reply: Thank you so much for such a valid and useful comment. We need to develop silage additives with different principles, which would control or modulate the fermentation pattern of the silages. We speculated that resin acids are one possibility, however they have not been tested as silages additives yet, and that is why we could not cite existing research in this area. Then, in order to express the importance of resin acids in this study, we added to the Introduction the following quote: “Resin acids are defence chemicals of coniferous trees, and it has been hypothesized that they could positively affect animal production, although effects on ruminants have not been proven. Because of their antimicrobial activity, resin acids may have potential to improve silage preservation quality and aerobic stability.”

 

Results and Discussion

1、L187-189 Emphasize the significance and winter hardiness of this grass as a perennial forage in cool and humid regions.

Reply: The following sentence regarding timothy characteristics is in Results and Discussion: “Timothy grass is the most important perennial forage grown for silage and hay production in cool humid regions of Northern Europe, Asia and North America. The popularity of timothy in these areas is mainly because of its winter hardiness.”

 

2、L209-212 Highlight the importance of low counts of molds and yeasts in the raw material during the harvesting period, as silage feed may be exposed to aerobic conditions during practical use.

Reply: The fourth paragraph of Results and Discussion section is specifically about the microbial quality of the raw material, including the reviewer’s request: “Low counts of moulds and yeasts in the raw material during harvest facilitate adequate aerobic stability of the silage when opened and exposed to aerobic conditions during the feeding period.”

 

3、L269-274 It is advisable to further explain the biological mechanisms behind the decrease in ammonia nitrogen concentration due to the effects of FPA, such as the impact of acidification on reducing protein breakdown.

Reply: Thanks for the comment. The biological mechanism of this process is clearly stated in the text. Please see text highlighted in yellow.

 

4、L276-279 Explain why even though ammonia nitrogen concentrations in C, HO, HE, and SA are higher than in FPA, they are still well-preserved.

Reply: Basically because the difference between those treatments is rather small, additionally according to Wilkinson (1990), grass silage with ammonia-N in the range of 50 – 100 g/kg total N are regarded as well fermented. This statement indicates that even if ammonia-N in C, HO, HE and SA was higher than in FPA, they were still well preserved. This information is clearly presented and highlighted in yellow in the text.

 

5、L280-285 Further discuss the lack of signs indicating the activity of ammonia-producing bacteria in the current material.

Reply: We stated in the text that even Control silages did not indicate the presence of clostridial fermentation, which would in turn produce ammonia. Thus, this was an intrinsic characteristic of the raw material utilized in this study. Thanks for the comment and this information is now highlighted in yellow in the text.

 

Final reply: Authors greatly appreciate comments and feedback from Reviewer #2 in improving our manuscript. Many thanks!

The authors.

Round 3

Reviewer 2 Report

General comments: This article evaluates the effects of different management options on silage quality through two experiments, including dry matter (DM) content, types of additives, and the impact of resin acid products on silage characteristics and aerobic stability. The article is forward-looking, however, there are some areas that require modification.

 

Introduction

1、L25 There is insufficient information about the influence of DM content on microbial activity, fermentation efficiency, and storage stability in the silage process. It is recommended to provide additional details in this regard.

 

2、L29 More background information can be provided regarding the potential impact of climate change on agricultural production and silage management.

 

3、L39 More information can be provided about the characteristics of resin acids, their role in the silage process, and existing research in this area.

 

Results and Discussion

1、L187-189 Emphasize the significance and winter hardiness of this grass as a perennial forage in cool and humid regions.

 

2、L209-212 Highlight the importance of low counts of molds and yeasts in the raw material during the harvesting period, as silage feed may be exposed to aerobic conditions during practical use.

 

3、L269-274 It is advisable to further explain the biological mechanisms behind the decrease in ammonia nitrogen concentration due to the effects of FPA, such as the impact of acidification on reducing protein breakdown.

 

4、L276-279 Explain why even though ammonia nitrogen concentrations in C, HO, HE, and SA are higher than in FPA, they are still well-preserved.

 

5、L280-285 Further discuss the lack of signs indicating the activity of ammonia-producing bacteria in the current material.

• Please read through the text to ensure fluency and accuracy