The Effects of Different Doses of 3-NOP on Ruminal Fermentation Parameters, Methane Production, and the Microbiota of Lambs In Vitro
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors1. The manuscript is about a ruminal fermentation with a simple experimental design. This study need to summarize or present new insights beyond those found in existing similar researches.
2. The experimental purpose is not well-defined, leading to a logical inconsistency in the manuscript. Many issues in the manuscript. For example, why can beef produce the methane in the same way as other ruminants in Line 45?
3. Merely explaining the meaning of the indicators without deeply interpreting the reasons behind significant differences is insufficient.
4. Please do not use any translation software to write the research paper. It’s hard to comprehend the writing, the manuscript requires a native English speaker to review and revise its grammar and sentence structure.
Comments on the Quality of English LanguageIt’s hard to comprehend the writing.
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for Authors
The authors conducted in vitro ruminal fermentations on the effect of three levels of the anti-methanogenic compound 3-NOP, using a ground total mixed ration as substrate and ruminal inocula obtained from lambs. They demonstrated some of the same effects as other workers observed using different inocula in vitro, or in vivo using other ruminants. There is not much originality to this work, but it does fill a small knowledge gap by extending the work to lamb inocula in vitro.
In general, the methods used were appropriate (aside from the unfortunate use of a kit-based community DNA isolation method), and the now-conventional analytical methods appear to have been competently performed. The reviewer’s primary concerns regard some unusual observations, the presentation format of some of the data, and the interpretation of some of the data.
One of the more surprising observations was the very small fraction of both the bacterial (1830/28601 = 6.4%, L280-281) and archaeal (339/1895 = 17.9%) communities that were shared across all four treatments, even though the treatments varied only in the level of a single ingredient. The reviewer is not aware of any ruminal community assessments in which so little commonality was observed. The authors should address this issue by comparing their results to those in other community assessments), and put forth some explanation as to why their treatments did not yield more commonality. They should also dissect their data a little more: For example, what percentage of the total ASVs did the shared group represent?
The authors should also do a better job of interpreting their microbial data with a view toward just how abundant the taxa are. One of their impressive results is the significance of Prevotella abundance on propionate, and Methanobrevibacter on methane production. So many reports in the literature make a big deal of a correlation between a given parameter and the relative abundance of a taxon that represents only a tiny fraction of the ruminal community (and thus seems unlikely to be a genuine effector of that parameter). Here, the authors show effects by the most dominant genus of bacteria (Prevotella) and of methanogen (Methanobrevibacter). This is worth explicit mention.
Specific comments:
L29-30: Clarification is needed here. Do the authors mean 0.05 mg per g of dietary DM (as opposed to, say 0.05 mg per lamb)? In other words, the authors need to be more specific in their units.
L89-90: Do the authors mean here that the lambs used as rumen fluid donors were fed 3-NOP in their rations?
L90: Pulverized how?
L95: There is a lack of clarity here. Was the strained rumen fluid obtained by combining the rumen fluid collected from all four lambs (L75)? Or were the separately donated rumen fluids used for separate incubations within run (i.e., two replicates per lamb donor within the 8 “replicates” within treatment (L104))?
L124: Why was this method of DNA extraction chosen? It is well known that kit-based DNA extractions give very low yields and highly non-representative community compositions from ruminal samples (Henderson et al., doi: 10.1371/journal.pone.0074787).
Table 2: The values are presented to an unreasonable level of precision. For example, one cannot measure gas production to the nearest microliter using a large syringe.
Table 3: Again, unreasonable levels of precision. Also, because the percentages for the four VFA listed total 100%, it is clear that the calculations exclude the branched chain VFA, This should be noted in the manuscript (either in the Methods, or the Results, or as a footnote in Table 3.
Figures 4 and 5: These bar graphs repeat the data in Table 4, to much weaker effect. Many of the bars are dominated by a single taxon with the minor taxa nearly (or completely) invisible within individual bars. The figures should be deleted, and the more useful Table retained, as it contains exact values including statistical comparisons among treatments.
L349: It’s not clear what the authors mean by “metabolization of fermentation products”. Do they mean fermentation product formation, or fermentation product utilization?
L361: Delete “rate”, a term which should be reserved for reaction kinetics. What the authors observed was a decrease in the extent of methane production, not the rate of methane production.
L361-366: This explanation is poorly written. On the one hand, it claims that the 97% reduction is consistent with similar reductions in other studies in which methanogen copy numbers were decreased, yet it this present study the methanogen copy number reduction was not statistically significant. So the “consistency” only involves methane production, not methanogen copy number.
L390-393: The logic for this conclusion is not apparent. 3-NOP affects methanogens after they have taken it up, and they affect methyl-CoM reductase, not methyl-CoM itself (i.e., 3-NOP does not react with methyl-CoM). So, how would uptake of methyl-CoM affect 3-NOP-mediated inhibition?
L413-415: This is an interesting observation, but what are we to make of it? Not all Prevotella produce propionate, and the reviewer knows of no reports in which H2 (whose concentration is elevated when methanogenesis is inhibited) serves as electron donor for propionate production.
L439-440: It is not clear what the authors are getting at. Are they claiming that, because protozoal cells are much larger, they have more intracellular volume to dilute any 3-NOP that was taken up, and this lower 3-NOP concentration would be less inhibitory to endosymbiotic methanogens?
L443-446: This statement as written seems a little silly. If the “former treatment” is the C-treatment (no 3-NOP), of course methane production would not be affected, because there was no treatment.
Minor edits:
L113-115: Rewrite sentence to indicate that the phosphoric acid was added to the culture subsample (as opposed to the culture having contained phosphoric acid).
L177: Change “liner” to “linear”.
L298-302, L325-342: Italicize all genus names.
L400: As archaea are also microorganisms, do the authors mean here “bacteria”, rather than “microorganisms”?
L419: Insert “metabolic” ahead of “hydrogen”.
L438-439: Change “changes” to “differences” (because the experiment only compared different treatments, it cannot detect “changes”).
Comments on the Quality of English Language
English is fine, only minor edits required as noted in Comments to Authors.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsI suggest some modifications
Abstract:
A definition of 3NOP is necessary, so I suggest that the abstract provide a brief introductory sentence.
How many repetitions per treatment did the authors use?
Were the analyzes performed in triplicate?
Keywords
Remove from the keywords those words contained in the title of the manuscript, as the title can be used to search for similar research, therefore, authors are suggested to insert new words in this item.
Introduction:
Lines 63 – 64: The authors state that those especially with lambs are scarce... but if they are scarce, they still exist, therefore, it is necessary to cite research that evaluated the object of the study and justify what innovation the current research presents in a way that has merit to be published.
A hypothesis must be inserted before the objectives.
Methodology
Line 76 - Enter the protocol number of the ethics committee for research using animals
Line 78 – a table with the chemical composition of the ingredients that make up the diet must be inserted.
Line 78: What is the roughage: concentrate ratio of the diet? The diet was formulated according to which recommendations? What is the estimated gain? How long did the animals receive the diet until their rumen flora was adapted, before collecting the rumen fluid?
Table 1 – Enter the values ​​of dry matter, ether extract, total carbohydrates, non-fiber carbohydrates, metabolizable energy and Crude protein / Metabolizable energy ratio of the diet
Line 92 - The in vitro gas production method needs to be rewritten again and more details and information I will suggest some references that may be useful to you
1- *Y.A. Soltan., A.S.Morsy., S.M.A. Sallam., H.Louvandini., A.L. Abdalla. 2012. Comparative in vitro evaluation of forage legumes (prosopis, acacia, atriplex, and leucaena) on ruminal fermentation and methanogenesis. Journal of animal and feed science, v.21.p.759-772 .2012. 2- Y. A. Soltan, A. L. Abdalla, L.R.F. Silva, A.S.Natel, A.S.Morsy, H.Louvandini. Response of different tropical pasture grass species to treatment with fibrolytic enzymes in terms of in vitro ruminal nutrient degradation and methanogenesis. Special issue of Animal Nutrition and Feed Technology v, 13: p 551-568, 2013. 3- A.S. Morsy, Y.A. Soltan, S.M.A. Sallam, M. Kreuzer, S. M. Alencar, A. L. Abdalla. Comparison of the in vitro efficiency of supplementary bee propolis of different origin in enhancing ruminal nutrient degradation and mitigating methane formation. Animal Feed Science and Technology. v,199: p 51-60, 2015. (DOI: 10.1016/j.anifeedsci.2014.11.004) 4- Y.A. Soltan A.S. Morsy R.C. Lucas A.L. Abdalla. Potential of mimosine of Leucaena leucocephala for modulating ruminal nutrient degradability and methanogenesis. Animal Feed Science and Technology. v, 223: P 30-41, 2017, http://dx.doi.org/10.1016/j.anifeedsci.2016.11.003 5- Y.A. Soltan, A.S. Natel, R.C. Araujo, A.S. Morsy, A.L. Abdalla. Progressive adaptation of sheep to a microencapsulated blend of essential oils: Ruminal fermentation, methane emission, nutrient digestibility, and microbial protein synthesis. Animal Feed Science and Technology. v, 237: P 8-18, 2018. https://doi.org/10.1016/j.anifeedsci.2018.01.004
Some figures are difficult to understand, I suggest the authors improve the resolution of the images and enlarge the font used.
Table 4 must contain information on probability values ​​in accordance with what was presented in previous tables
Author Response
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Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
The authors have addressed essentially all of the reviewer’s concerns, and thus have greatly improved their manuscript. Only a few minor issues remain:
L69: Delete “studies on”.
Table 3: The authors have expressed gas volume at a reasonable level of precision, but the values for other values in the table are still unreasonably precise by at least an order of magnitude.
Table 4: Again, an unreasonable level of precision. Round to nearest 0.01.
L529: Insert “for” after “Unlike”.
Author Response
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Author Response File: Author Response.pdf
