Effect of Formic Acid and Inoculants on Microbial Community and Fermentation Profile of Wilted or Un-Wilted Italian Ryegrass Silages during Ensiling and Aerobic Exposure
Round 1
Reviewer 1 Report (New Reviewer)
The research carried out by the authors is complex and properly conceptualized. The practical importance of the research results in obtaining high quality silages is highlighted. The aim of this research work is well defined. The authors presented the issues of originality through the analysis of some research on the similar theme.
Research methodology is presented in detail, designed in well-organized subsections. The results are exposed and analyzed in depth, being supported by clear, eloquent images.
Conclusions are concise. It would be advisable for the authors to complete the conclusions with some information about the valorization of the results, especially mentioning practical applications and sectors (economic, academic) that can benefit from these results.
Bibliography is relevant but quite old, I recommend supplementing the introduction with information citing current references.
There are some mistakes in expressing ideas and editing the text. Therefore, I expect the authors to correct the following errors:
1. The first two sentences in the Abstract do not contain a verb, please rephrase.
2. Indicate the meaning of P, in the abstract but also when it first appears in the text.
3. Line 42: please replace <compare> with <compared>, or reformulate.
4. Line 52: please replace <increased> with <increase>, or reformulate.
5. Paragraph 153-159 is a text taken ad-literam from the procedure, please adapt it to the past tense.
6. More attention to the use of Italic in naming the species. See the lines 277, 280, 308, 358, 373, 394, 395, 402.
7. Line 281: please replace <are belong> with <belong>
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report (New Reviewer)
Review report
Fermentation
fermentation-1949012
Effect of formic acid and inoculants on microbial community and fermentation profile of wilted or un-wilted Italian ryegrass silages during ensiling and aerobic exposure
Qifan Ran1† , Hao Guan2† , Haiping Li3 , Wei He1 , Ruifen Zhu1 , Yong Huang1 , Yuandong Xu1* and Yan Fan1 *
1 Institute of Grassland Science, Chongqing Academy of Animal Sciences, Chongqing 402460, China; 6 [email protected]
2 Sichuan Zoige Alpine Wetland Ecosystem National Observation and Research Station, Southwest Minzu University, Chengdu 610041, China; [email protected]
3 School of Mathematics and Statistics, Qinghai Normal University, Xining 810016, China; 10 [email protected] 11 12
* Correspondence: [email protected]; [email protected]
† Authors contributed equally to this article and are co-first authors
Summary Rating
Originality: fair
Technical Quality: fair
Clarity of Presentation: fair
Importance to field: fair
Evaluation:
This manuscript will be acceptable after major revisions
Comments:
The manuscript aims to investigate the influence of L. plantarum, L. buchneri and formic acid microbial community and fermentation profile of wilt or un-wilt Italian ryegrass silages during ensiling and aerobic exposure.
The authors concluded that the DM content of Italian ryegrass affected its epiphytic microbial community and the effectiveness of the different type of additives. Formic acid was more suitable for un-wilted Italian ryegrass silage, L. plantarum had a better effect in wilted Italian ryegrass silage, and L. buchneri prolonged the aerobic stability of Italian ryegrass. DM content and purpose of ensiling should be the key factors for choosing different type of additives for Italian ryegrass silage.
Although the originality is not high. The work is useful in feed science and ruminant nutrition area. The technical quality and clarity of presentation are fair.
But there is a big issue in experimental design and data analysis.
I recommend that this manuscript will be acceptable after major revision.
Please see the following for my suggestions to be considered:
• L74-79: Author state “Italian ryegrass (Lolium multiflorum Lam. ver. Chang Jiang II) was grown in the farm of the Chongqing Academy of Animal Sciences (N 29.32°, E 105.59°, 510 m above sea content). The grass was harvested from three different fields at the heading stage on 27 April 2019. All of fresh forage were well-mixed and separate equally into two parts, half of the fresh grass (around 25% fresh weight) were directed chopped and another half of fresh grass were dried with nature wind in shadow until the DM was around 35% of fresh weight (FW)….”: Question: if all samples were mixed, half for fresh treatment, the other half for dried treatment. What are your real replications in your experiments? You cannot use subsamples from each treatment (fresh sample or dried sample) as your replications.
• L186-189: Table 1. What are your replications? add n=? and SD value if you have real replications (n value and replication should NOT be from your subsamples !!!)
• L237-242: Table 3. There is an issue in presenting statistical analysis results and misunderstand the statistical results. For example, DMR: interaction is not significant, how can you give letters for all treatments? (DMR: 97.29c 98.71ab 97.00c 96.83c 97.90bc 99.21a 97.00c 97.54bc 0.39 * * NS).
• L173-181: Please add your analysis model. If you CRD, please add CRD model assumption checking. Which method did you used to check CRD assumption? Clearly indicate whether model assumptions meet or not.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report (New Reviewer)
This manuscript investigated how the microbial population and fermentation profile of wilt or un-wilt Italian ryegrass silages were affected by L. plantarum, L. buchneri, and formic acid during ensiling and aerobic exposure. The subject itself is surely worthy of investigation. However, some points need to be addressed as follows:
1) Lines 62-64: “Ensiling is a fermentation ……. change drastically” please revise the sentence meaning.
2) The hypothesis of the study should be clarified before the aim at the end of the Introduction section.
3) Line 79: “….until the DM was around 35%...” How long did that process take?
4) Line 80: How was the grass chopped?
5) Line 82: What is the concentration of formic acid?
6) The authors had to justify on what basis they selected the levels of the tested additives.
7) Provide full details of all additives/products used, ie, product, concentration, company, city, country).
8) Lines 103-105: Correct the order of citations (17, then 18, and 19).
9) Line 104: update citation used for CP analysis.
10) Lines 106-115: Add a reference for the microbial analysis.
11) Lines 116-121: Insufficient description of fatty acid profile method; add standard substances with catalog numbers. How were FAs identified? E.g. using a standard mixture of fatty acids.
12) Line 130: Silage temperature data are not mentioned in the results section. Please add.
13) It is highly recommended to transfer Table 1 to the M&Ms section.
14) Tables 2-4: Add the exact p-value instead of using ** or NS.
15) Table 2: The pH values in the FA groups were lower than the control, however, the lactic acid concentrations were lower than the control. The pH value is inversely correlated with the lactic acid content. Please revise your raw data and correct it.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report (New Reviewer)
No further comments are to be addressed.
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
Manuscript ID: fermentation-1648146
Effect of formic acid and inoculants on microbial community and fermentation profile of wilted or un-wilted Italian ryegrass silages during ensiling and aerobic exposure
by Ran et al.
General comments
The objective of the manuscript proposed by the authors could have some interesting new information, mainly in relation with the comparison of the microbial inoculant to the chemical additive. The impact of chemical additives on the silage microbiota had been less studied.
In the last three years, the number of publications related to understanding changes to the microbiota during ensiling was very important, with around 100 new publications. The authors basically ignored most of those publications, both in the introduction and in the discussion. This is generally inconceivable. The authors also ignored to discuss a paper by Ni, et al (DOI: 10.1007/s00253-016-7900-2) performing a similar PCR-DGGE analysis of Italian ryegrass. They also ignored all previous PCR-DGGE analysis of silage previously published (for review DOI: 10.3168/jds.2017-13704).
In relation with the experimental design, the number of repetitions (3) performed by the authors was at the limit of what should be expected in silage research. Considering the time needed to prepare the trial, wilt the forage, etc. you will gain in statistical power by increasing the number of repetitions (five should be a good target).
Lastly, I do not understand why the PCR-DGGE analysis was performed from only one set of repetition. If more repetition were performed, it would then be important to change to the way the analysis was performed and possibly report the mean of presence/absence for individual bands.
The authors should expand their discussion to explain why they performed the trial using PCR-DGGE instead of amplicons sequencing using next generation sequencing. PCR-DGGE still have some potential in relation to the target specific strains of a genus, but not for a diversity study. Considering the cost of performing amplicons sequencing actually, PCR-DGGE and the needs to clone and sequence to identify the bands offer no advantage for a general diversity study.
Specific comments
Major change throughout the text:
- All scientific names (genus species) should be formatted to italic
- All subscript or superscript needs to correctly edited in the text
- Remove dash in words (example in line 52, 54, 55, 57, 59, continue…)
Lines 168-170: Remove this section as this is from the model document
No need for Table 1 – all the data was detailed in the text (lines 173-177).
Line 223: Not significant in the table. Modify the text
Line 257 : Replace “heated” by “heating” or “stable”
Idem for line 259
All figures, do not split in two paragraphs, melt into one.
Line 275: should belong to the family Bacillaceae instead of the genus Bacillus.
Line 360: Yeast were not detected (ND) in FA samples, how can you state this.
Line 385: add citation at the end of the sentence
Line 392: add citation at the end of the sentence
Line 395: add citation at the end of the sentence. Also explain why.
Line 398: add citation at the end of the sentence (with older articles on formic acid)
Line 403: information on Bacillus during aerobic exposure is contrary to published results. Please explain.
Line 407: replace “degeneration” by “deterioration.”
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
The manuscript 1648146, entitled “Effect of formic acid and inoculants on microbial community and fermentation profile of wilted or un-wilted Italian ryegrass silages during ensiling and aerobic exposure” suffers from less-than-adequate use of English language. Moreover, there are design limitations that must be explained and discussed and above all, it lacks originality; the authors make no such claim, anyway. Conclusions are correct but already known. Research in Europe has shown these results 30-40 years ago. The authors must make an effort to highlight the novelties of their research.
Specific comments follow:
ABSTRACT
L15-29: In a way, the abstract is misleading regarding the Materials and Methods used, see comments in relevant section.
INTRODUCTION
L33: “short-lived”, awkward selection of words.
L35: good nutritional quality.
L36-37: can be fed as fresh forage mainly…. consider moving the rest in the beginning of the sentence.
L39: what seasonal imbalances?
L40: There is no method to improve the feeding value of fresh forage, please rephrase, you probably mean that silage is better than hay.
L41: Replace “preparation and storage” with “production”.
L43-44: “Italian ryegrass is not easy to produce valuable silage”. Please rephrase.
L44: “In addition” not needed, see L42.
L47-49: How do you increase dry matter content for ensiling other than wilting?
L48: “effects” not “influences”.
L49-50: “raw materials” or fresh forage? And…and….and… Please rephrase.
L52: “affect microflora” How?
L53: how much high?
L56: “besides” may not be the right choice, here.
L61-67: Please avoid repetitions, incorporate whatever needed in previous lines’ text. Do “microorganisms change drastically”? What is the meaning of this?
MATERIALS & METHODS
L72-86: Reading this part of the text, I realized for the first time that this was solely a "lab experiment". Neither the title of the manuscript nor the abstract gave any opposite indication before. With "lab experiment", I mean that all work regarding sample preservation etc was done in the lab and samples were not left exposed under barn conditions as part of the silo fermentation or feedbunk exposure etc. However, this is important to know from the very beginning when someone reviews or just reads this paper. Proper adjustments in the title and abstract are needed. Moreover, information on material selected is practically absent; what do you mean by "part" in L74 and L75? What was the initial amount collected, was it mixed before dividing it in two "parts", how was one of them wilted and so on. "Piles" in L81 imply that a large quantity was originally used, is that true? If data or references that "lab" results are similar to "silo/feedbunk" results must be provided. Above all, is the number of replicates (3 for each treatment) large enough? How was this calculated?
L74: In view of the chemical analysis, please explain the "heading" stage with more details.
L87-121: This reviewer has no knowledge whether the procedure used is standardized regarding fresh and silage forage samples. References provided, cover the final chemical, fermentation etc analysis stages. Have all these procedures been validated? Are there references? See for example L124 (Guan et al. [14]).
L89-90: "ensiled silages"?
L161-167: A better and more detailed description is needed. "DM content" is confusing when used to describe wilted and non-wilted samples.
L167-170: Please, delete text starting from "3. Results.............".
RESULTS
L174: Use the word "fresh" here.
L175-176: Compared to European values (INRA 2007), NDF and especially ADF values appear rather high. Can you elaborate on that?
L185-187: While FA had the lower pH and lactic acid concentration.
L188-189: "while the control and LB-treated group at 25% DM were higher than at 35% DM". Please check again if this is true.
L199: I do not understand this sentence.
L201-202: Please check again if this is true. And it can't be Table 3.
L223-225: If I am not mistaken, dry matter recovery was not mentioned or explained in the Materials and Methods Section. Please check again if this is true.
L234-237: Certainly, one sees much more regarding NDF and ADF, when studying Table 3. Especially when taking into consideration the chemical analysis of fresh forage.
L240-241: Compared with the silages "at bag opening". The same applies in L246.
L246-247: That is not true for FA-treated silage.
L257-261: Text needs to be completely revised to make any sense. Choose your words carefully , please.
L268-323: English language and syntax must be improved.
DISCUSSION
L337-338: You need references regarding the DM content and the LAB number issues.
L339: "modulate"?
L361-362: This sentence is difficult to follow.
L363-411: English language and syntax must be improved.
CONCLUSIONS
L416-417: This sentence is difficult to follow.
TABLES
- a) Superscripts are used in a way I can't follow.
- b) *** symbols must be explained.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The new version of the manuscript provide important improvements over the previous versions (updated citations), but my key comments were not answered. Most of these are inherent to the design of the trial and could not be improved at this stage of the project.
- Low number of repetition
- Only one sample per treatment for the community analysis.
This new version is full of typing errors. Manuscript need to be completely checked. Example line 252 with a "t" floating alone in the text or line 206 with "-1" not in superscript.
Reviewer 2 Report
Two major comments:
1. Still, extensive editing of English language and style required.
2. Number of "repetitions" (3) too low for a valid research.
