Comparative Analysis of Wheat Hay and Silage in Methane Production, Fermentation Characteristics and Microbiota Using In Vitro Rumen Cultures

Round 1

Reviewer 1 Report

The article entitled “Comparative analysis of wheat hay and silage in methane production, fermentation characteristics and microbiota using in vitro rumen cultures” has some inadequacy of the information that should be corrected before publication. The following points need to be corrected and improved:

 

1. In the “Introduction” – The significance of the study needs to be explained clearly in this section. Is the in vitro study enough to justify the microbial diversity in the rumen ecosystem? Is there any consequence of in vivo study necessary for better understanding of the microbial ecosystem in relation to the mentioned forages?
2. In the “Materials and Method” Line 111 & 112: “The buffered medium for incubation was prepared according to the previous study [16]” – Please explain shortly the in vitro buffer medium. What is the ratio of Rumen fluid: Buffer mixture in the incubation?
3. In the “Materials and Method” Line 130 to 133: In table 2, Total gas production, CO₂ & CH₄ are included. Please briefly describe the methodologies of these parameters with references.
4. In the “Materials and Method”; Please include the protozoa count method and protozoal count result in Table 2.
5. In the “Result” Section Table 2: Please provide the Footnotes in the Table 2 for “MW”, “MWS”………”DWS15” as Table 1.
6. In the “Discussion” Section, line 319-321: Please provide the reference [The greater gas production in the wheat, which harvested at 15:00 (MWH15, MWS15, DWH15, DWS15) may because that the enzyme activity evaluated or some harmful bacteria were killed through the strong sunshine at noon,…..]
7. Please discuss little bit in the discussion section, why the A:P differed significantly, although no significant differences occurred in acetate or propionate production in the in vitro study?
8. In the “Discussion” Section, line 329-331: ..[It was also possible that the number of methanogens and protozoa reduced in the rumen fermentation process after the wheat was dried.]. why? Please justify your statement with reference.
9. In the “Discussion” Section, line 328-341: Please mentioned about the changes of protozoa in different experimental diets in this paragraph. Then discuss the relationship between methanogens and protozoa, and their influence on methane production with references.
10. In the “Discussion” Section: Please discuss little bit that no concentrate was used in the in vitro study as substrate. Only different types of forages were used. Using forage without supplementation of concentrate causes fewer proteolytic bacteria.
11. Please review the article for English. A huge mistake in the use of articles (a/an/the) in the manuscript. Please edit the manuscript for the English language!

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript reports the effects of wheat hay and silage on ruminal methane emission, fermentation, and microbial ecology in vitro. This study is crucial since authors clarified the changes in ruminal environment associated with two different forms of feeding wheat in ruminanta. The manuscript is well-written and the methods section is clear. However, the few following comments to the authors need to address before the manuscript can be considered for publication.

L38: Define NO, CO, and PM 2.5

L45: Describe the changes in fermentation

L53: Clarify the benefits on methane mitigation

L64: Here and throughout the manuscript, stick to microbiota rather than microbiome

L69: Here and throughout the manuscript, stick to whole-crop wheat

L70: Why?

Table 1: Define “a” in “aNDF"

L104: Cows or bulls?

L113: Describe briefly

L147: Add the kit brand name

Table 2: Define all the expressions under “P-value” section

 

 

 

 

 

 

 

 

 

 

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

The statistical model used in the study is correct, but I think the report of results can be substantially improved by dropping the interactions from the model, focusing exclusively in the main effects. Unless authors have actually hypothesized that interactions were expected to be non-significant (a non-significant interaction means something in this case and it helps to evaluate the hypothesis), it is better to drop them from the model. A more simplified model will provide a more clear idea on the main effects and will make tables and results much easier to report and for the reader to understand.

L38: check NO and CO are the main gases released from combustion of Straw.

L48-49: although methane is released from enteric fermentation, it is too stereotypic to affirm that ruminants are a “big environmental issue”. This is a matter of controversy. I suggest to suppress this statement.

L56: it is possible that this sort of nutritional strategies may contribute to reduce methane production, but it is too pretentious to say that it can suppress global warming and climate change. Authors muct be more moderate regarding these statements.

L59: translating is not an appropriate word here

L60-63: rewrite, not sure what is the meaning of this sentence

L67 replace “variance” with “variation”

L69-71 explain why

L74 replace “treated” with “preserved”

L82 replace “was dried by exposure to the sun” by “was sun cured (hay)”. Importat, state clearly the times for hay-making and for ensiling

L84-89: always replace “treated into” with “preserved as”

L90 replace “fermentation substrate” with “preserved forages”

L96-101: always replace “treated into” with “preserved as” (also in all the other table footnotes)

Table 1: use ANOVA to examine the effects of the factors on chemical composition. I would say that interactions can be omitted.

L112: rewrite “has continuous access”? (wrong expression)

L112 replace “0.2000 g ground feed substrate” with “Ground feed substrate (200 mg)” and say how fine it was ground

Vansoest is Van Soest

L135: why 3 aliquots and was microbial composition determined in each of these aliquots?

L139: why 6 aliquots and were ammonia and VFA determined in each of these aliquots?

L147: nnot clear whether a “culture sample” is a blend of 3 aliquots (1 ml each) of if it is each aliquot?

L184-187: I have seen that double interactions were in almost all cases non-significant. Moreover, in these designs, the triple interaction is very difficult for interpretation and in most cases it was also non-significant. I suggest a much more simple design in which all non-significant interactions are suppressed in the model, thus focusing only on the main factors. This would be much more clear for the reader to understand the main effects.

Results section: it would be much easier to follow if authors just focus on the main effects, and describe interactions only when significant and with biological relevance

Section 3.3. it would be much better to explain the main effects (hay vs. silage; morning vs. afternoon harvest, milk vs. dough stage). For instance in L254, how can you make an interpretation of the comparison of DWS15 group vs MWS7?

L290-291 most of these taxa are not genera? (Rikenellaceae, Christensenellaceae or Ruminococcaceae)

Methanobrevibacter are not bacteria but archaea, how could you measure them if you used bacterial primers?

L311-314: not relevant, a strong buffer is used for the incubations

Focusing only on main effects would also benefit the drawing of more clear and relevant conclusions

The list of references requires a thorough revision to fit the journal guidelines for the format of references (journal names sometimes in full sometimes in short, article title sometimes in lower case sometimes in capitals, etc. there are lots of inconsistencies and mistakes)

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Two minor issues for the revised manuscript:

1) State clearly the time required for hay-making (time forage was exposed to sun for drying) and for ensiling (days from filling the silos to opening them)

2) Dropping the interactions is not just to omit them in the Tables. The ANOVAS need to be re-run including only main effects (and not teh interactions) in the model, so the significance of the main effects may change compared with the full model. Also, when reported in Tables or Figures there is no point to show the 8 mean values (these are reflecting the interactions) but just the average of hay vs. that of silage; average of morning vs. that for afternoon harvest, and average of milk vs. dough stage. I suggest to change this, at least in the tables, and revise all the P-values

Author Response

Please see the attachment. Thanks a lot.

Author Response File: Author Response.pdf