Effects of Lactic Acid Bacteria Inoculants and Stage-Increased Storage Temperature on Silage Fermentation of Oat on the Qinghai–Tibet Plateau
Round 1
Reviewer 1 Report
This paper investigated the effects on the fermentation characteristics and chemical composition of oat silage from the Qinghai-Tibet Plateau.
I think that very meaningful data is shown, but there are many elements to consider, and I made it as simple as possible, for example, the relationship between the type of added microorganisms and staged storage temperatures, etc. I felt it was better to go to
In order to proceed with the current situation, I would like you to consider the following points.
P2L71- 2. Materials and Methods
　2.1 Silage preparation
The type and size (volume, etc.) of the silos used in the oat silage preparation should be indicated.
It should also indicate the packing density of the oats.
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　2.4 Statistical analysis
　Statistical processing is One-Way-ANOVA, but the main effects are considered to be LAB Inoculations (A), Staged storage temperatures (T), and Staged storage time (D). For this reason, it is an aspect of a multi-way analysis of variance, and it is questionable whether the application of One-Way-ANOVA is optimal. Why don't you try to make it simpler and consider it as I gave earlier?
In addition, Duncan analysis is used for multiple testing. There is some debate about the method of multiple testing, but it has been pointed out that the results of Duncan analysis are naive. Isn't it necessary to investigate using more advanced analysis methods such as those of Tuky and Fisher?
Author Response
We appreciate for Reviewers’ warm work earnestly. According to the reviewers’ comments on our manuscript “Effects of lactic acid bacteria inoculants and stage-increased storage temperature on silage fermentation of oat on the Qing-hai Tibetan Plateau” (fermentation-1986246), we have made modifications to our manuscript. The following is the response to the reviewers’ comments.
Point 1: The type and size (volume, etc.) of the silos used in the oat silage preparation should be indicated.It should also indicate the packing density of the oats.
Response 1: Thank you for your suggestion! We have added relevant information to the corresponding position of 2.1 silage preparation in the manuscript.
Point 2: Statistical processing is One-Way-ANOVA, but the main effects are considered to be LAB Inoculations (A), Staged storage temperatures (T), and Staged storage time (D). For this reason, it is an aspect of a multi-way analysis of variance, and it is questionable whether the application of One-Way-ANOVA is optimal. Why don't you try to make it simpler and consider it as I gave earlier?
In addition, Duncan analysis is used for multiple testing. There is some debate about the method of multiple testing, but it has been pointed out that the results of Duncan analysis are naive. Isn't it necessary to investigate using more advanced analysis methods such as those of Tuky and Fisher?
Response 2: Thank you for your suggestion! We also think that the table processing is not appropriate. This tables are redundant and difficult to understand. In addition, the figures have fully expressed and confirmed our views. Based on your suggestion, we deleted these tables.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Manuscript ID: Fermentation 1986246
Effects of lactic acid bacteria inoculants and stage-increased storage temperature on silage fermentation of oat on the Qing-hai Tibetan Plateau
By L Li et al.
General comments
The objective of the trial described in the manuscript was to understand the role of a silage inoculant and an increase in incubation temperature on the quality of oat silage.
The trial would have brought valuable information on the impact of low ambient temperature during incubation or storage of silage.
Still important methodological issues are present in the manuscript. The main one is that the authors quantified the fermentation products of the silage using a methodology for ruminal fluid (citation to Broderick and Kang, 1980). This is clearly not the way that fermentation products are analyzed in silage.
This renders the preparation of all four tables of the manuscript absolute. Only need one mainly. Also include the units for the different parameters included in the tables.
I also to not understand how the computation was made for the different parameters included in the tables. It is quite puzzling to see pH values of -80.50 and 86.29 (Table 2) or of 21.98 and of 32.02!!!!!! (Table 4)
The authors should also:
- Improve the description of the “stage increased” of the temperature since the corresponding description and preparation of the main tables is really hard to follow. This should be explained in the introduction as well as in the methodology. A recall should be used in the Results and Discussion section
- How did you compute the “lag time”. Describe in methodology
- Use p value “< 0.001” instead of 0.000 in the manuscript and the tables
A section of the methodology is describing Illumina MiSeq amplicons sequencing. No steps for DNA extraction were described. No clear used of this methodology appears in the Results and Discussion section.
Important issues related to the English language used and for the typography.
Specific comments
- Always use a space before opening a parenthesis (see line 13, 14, and several places afterward in the manuscript)
- Always use a space before opening a bracket (see line 49 and several places in the manuscript)
- Format citation according to guidelines. When using the form “Bai et al. (2022) [8]” (see line 53 and multiple places in the manuscript), you do not need to insert the year. Should be “Bai et al. [8]” or “Bai et al. (2022)”. Check guidelines.
- Use the past time when referring to the trial. See line 73 (“is”) and throughout the manuscript.
- Scientific name should be “L. buchneri” with lower case letter for the specie. See line 80 and throughout the manuscript.
- Idem for the genus name, which should start with an upper case letter (see line 230). Check manuscript. Also should update nomenclature to Lactiplantibacillus.
- Lines 73-74: I do not understand the unit used for the location of the experimental site)
- Line 88: replace “stomatal” by “repiprocal”
Author Response
We appreciate for Reviewers’ warm work earnestly. According to the reviewers’ comments on our manuscript “Effects of lactic acid bacteria inoculants and stage-increased storage temperature on silage fermentation of oat on the Qing-hai Tibetan Plateau” (fermentation-1986246), we have made modifications to our manuscript. The following is the response to the reviewers’ comments.
Point 1: Still important methodological issues are present in the manuscript. The main one is that the authors quantified the fermentation products of the silage using a methodology for ruminal fluid (citation to Broderick and Kang, 1980). This is clearly not the way that fermentation products are analyzed in silage.
This renders the preparation of all four tables of the manuscript absolute. Only need one mainly. Also include the units for the different parameters included in the tables.
I also to not understand how the computation was made for the different parameters included in the tables. It is quite puzzling to see pH values of -80.50 and 86.29 (Table 2) or of 21.98 and of 32.02!!!!!! (Table 4)
Response 1: Thank you for your suggestion. We also think this method is not suitable. According to your suggestion, we deleted these tables. In addition, the figures have fully expressed and confirmed our views.
Point 2: Improve the description of the “stage increased” of the temperature since the corresponding description and preparation of the main tables is really hard to follow. This should be explained in the introduction as well as in the methodology. A recall should be used in the Results and Discussion section
Response 2: Thank you for your suggestion! According to your suggestion, we have further explained in the introduction and methodology of the manuscript, and used a recall in the Results and Discussion section.
Point 3: A section of the methodology is describing Illumina MiSeq amplicons sequencing. No steps for DNA extraction were described. No clear used of this methodology appears in the Results and Discussion section.
Response 3: Thank you for your suggestion! We used Quantitative real-time PCR technology.
Point 4: Always use a space before opening a parenthesis (see line 13, 14, and several places afterward in the manuscript).
Response 4: Thank you for your suggestion! We have corrected the mistakes in the manuscript, and we are very sorry for such mistakes.
Point 5: Always use a space before opening a bracket (see line 49 and several places in the manuscript).
Response 5: Thank you for your suggestion! We have corrected the mistakes in the manuscript, and we are very sorry for such mistakes.
Point 6: Format citation according to guidelines. When using the form “Bai et al. (2022) [8]” (see line 53 and multiple places in the manuscript), you do not need to insert the year. Should be “Bai et al. [8]” or “Bai et al. (2022)”. Check guidelines.
Response 6: Thank you for your suggestion! We have corrected the mistakes in the manuscript, and we are very sorry for such mistakes.
Point 7: Use the past time when referring to the trial. See line 73 (“is”) and throughout the manuscript.
Response 7: Thank you for your suggestion! We have corrected the mistakes in the manuscript, and we are very sorry for such mistakes.
Point 8: Scientific name should be “L. buchneri” with lower case letter for the specie. See line 80 and throughout the manuscript.
Idem for the genus name, which should start with an upper case letter (see line 230). Check manuscript. Also should update nomenclature to Lactiplantibacillus.
Response 8: Thank you for your suggestion! We have updated nomenclature to Lactiplantibacillus, and we are very sorry for such mistakes.
Point 9: Lines 73-74: I do not understand the unit used for the location of the experimental site.
Response 9: The experiment base at the Sichuan Academy of Grassland Science, which is located on the southeast edge of Qinghai-Tibetan Plateau (Hongyuan; N31°51 '-33°33', E101°51 '-103°22'; altitude 3500 m).
Point 10: Line 88: replace “stomatal” by “repiprocal”
Response 10: Thank you for your suggestion! We have replaced the word in the corresponding position of the manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
I confirmed the correction of the content.
The content was devoted to grasping the phenomenon of the effects of additives, and I think it was easier to read than the papers submitted earlier.
I felt that it would be a knowledge about solving local problems.
Author Response
Thank you for your suggestion! It's a great honor to get your comments.
Author Response File: Author Response.docx
Reviewer 2 Report
Manuscript ID: Fermentation 1986246 Revision 1
Effects of lactic acid bacteria inoculants and stage-increased storage temperature on silage fermentation of oat on the Qing-hai Tibetan Plateau
By L Li et al.
General comments
Results of silage fermentation performed under low temperature conditions are not frequent and additional data is always welcome. The results from this manuscript are good improvements are required.
Additional comments for R1
By removing the four tables present in the initial version of the manuscript, the authors are now able to allow the reader to concentrate on the figures showing the changes in the different parameters associated with the “stage-increased in storage temperature” performed.
In the methodology, it will be important to improve the description of the “stage increased”. This should be explained in the introduction as well as in the methodology. A recall should be used in the Results and Discussion section. I am not sure if the authors performed an increase of the temperature to 10 and 15°C or by 10 and 15°C. Take the time to write this out using additional details. The authors are mentioning at lines 92-94 that the silos were stored at ambient temperature varying between 2 to 20°C. How does the “stage increased” fit here?
> Possibly prepare a figure describing what has been done and the link with the ambient temperature. This should include that link with the time (5, 10, 15 d) as there is another time of storage involved somewhere…
I am not certain for this, but I think that “stage increased” should be written “staged increase” instead.
In my first comments, I mentioned that “A section of the methodology is describing Illumina MiSeq amplicons sequencing. No steps for DNA extraction were described. No clear used of this methodology appears in the Results and Discussion section.”
In their reply, the authors mentioned that a qPCR was performed, and they added the details for the DNA extraction. This is okay, but we still do not know what they intend to quantify using the qPCR and details of an Illumina sequencing and bioinformatics manipulations are still present in the methodology.
> I was not able to find the data of a quantification PCR in the results.
The statistical analysis section needs to be expanded. In the revised manuscript, we have no details on the statistical model used for the ANOVA. The authors could also perform time-based analysis to
No statistical results (p values) are mentioned directly in the text or the figures. The figures include a deviation mark that is identified as SEM but is quite possibly the standard deviation considering the difference in size it as between treatments. This need to be addressed.
The conclusion is good, but will have to refer to updated statistical results.
Some issues related to the English language used and for the typography.
Specific comments
- Line 20: Do not delete the full name of the genus here. Keep Lactiplantibacillus.
- Line 69: replaced to “…a phased increased by 10 °C and 15 °C was used during storage to explore…”.
- Line 70: “silage” with lower-case S.
- Line 74: lower case for “oat” and “stage”.
- Line 81: again, are you sure of the position. The details are mentioning 2 degrees spread North-South as well as East-West for the area. This is very important. The forage was quite possibly harvested on a more precise location.
- Line 85: replace “Lactobacillus plantarum” by “Lactiplantibacillus plantarum”.
- Line 87: change “CFU/G” to “CFU/g” and define FM.
- Line 88: idem to 87.
- Line 88: replace “and Provided Gaofuji” by “and provided by Gaofuji”.
- Line 91: it could be a good idea to use an acronym that the reader could remember for the “ambient temperature”. We have no idea what “ZF” is standing for.
- Line 92-94: How did this step relate to the ambient temperature is linked to the stage increased?
- Line 107: “agar” should be lower case.
- Line 156: replace “obtained the same results” by “obtained similar results”. The experimental conditions were different, so it is only possible to say that the trends were similar.
- Line 204: add correct figure numbers (may require checking throughout the text).
- Line 211: replace “ ; ” by a dot ( .).
- Line 226 and 253: in this case, you can use the present tense (“oat silage is shown in Figure 3”) since this refers to the manuscript and not the results of the trial.
- Line 227: should include a citation for the link to the pungent smell and palatability.
- Line 228: not clear “can make the silage of feed more excellent”.
- Line 260: replace “…was higher, our research has similar results.” by “was higher. The results of the 10°C staged increased show similar results.”
- Line 271: replace “ ;” by an end of sentence dot (before “When”).
- Line 271: what do you mean by “number of attached colonies…” ? Are you referring to the cell counts of the inoculant or to the cell count of the epiphytic lactic acid bacteria?
Author Response
We appreciate for Reviewers’ warm work earnestly. According to the reviewers’ comments on our manuscript “Effects of lactic acid bacteria inoculants and stage-increased storage temperature on silage fermentation of oat on the Qing-hai Tibetan Plateau” (fermentation-1986246), we have made modifications to our manuscript. The following is the response to the reviewers’ comments.
Point 1: In the methodology, it will be important to improve the description of the “stage increased”. This should be explained in the introduction as well as in the methodology. A recall should be used in the Results and Discussion section. I am not sure if the authors performed an increase of the temperature to 10 and 15°C or by 10 and 15°C. Take the time to write this out using additional details. The authors are mentioning at lines 92-94 that the silos were stored at ambient temperature varying between 2 to 20°C. How does the “stage increased” fit here?
> Possibly prepare a figure describing what has been done and the link with the ambient temperature. This should include that link with the time (5, 10, 15 d) as there is another time of storage involved somewhere…
I am not certain for this, but I think that “stage increased” should be written “staged increase” instead.
Response 1: Thank you for your suggestion! We have added relevant information in the corresponding position of the manuscript. We put them in the incubator (10°C and 15°C) for 5, 10 and 15 days. Then take it out and stored at ambient temperature (2-20 °C) for 5, 10, 15, 30 and 60 of ensiling, respectively.
Point 2: In my first comments, I mentioned that “A section of the methodology is describing Illumina MiSeq amplicons sequencing. No steps for DNA extraction were described. No clear used of this methodology appears in the Results and Discussion section.”
In their reply, the authors mentioned that a qPCR was performed, and they added the details for the DNA extraction. This is okay, but we still do not know what they intend to quantify using the qPCR and details of an Illumina sequencing and bioinformatics manipulations are still present in the methodology.
> I was not able to find the data of a quantification PCR in the results.
Response 2: Thank you for your suggestion! QPCR was used to calculate the number of microorganisms (LAB and L. plantarum).
Point 3: The statistical analysis section needs to be expanded. In the revised manuscript, we have no details on the statistical model used for the ANOVA. The authors could also perform time-based analysis to No statistical results (p values) are mentioned directly in the text or the figures. The figures include a deviation mark that is identified as SEM but is quite possibly the standard deviation considering the difference in size it as between treatments. This need to be addressed.
Response 3: Thank you for your suggestion! The figures are based on SEM values.
Point 4: Specific comments
Response 4: Thank you for your suggestion! We have corrected the mistakes in the manuscript, and we are very sorry for such mistakes.
Author Response File: Author Response.docx
