Untargeted Metabolomics Reveals Intestinal Pathogenesis and Self-Repair in Rabbits Fed an Antibiotic-Free Diet

Round 1

Reviewer 1 Report

The authors have modified the manuscript according to the recommendations made, which has improved the manuscript. However, there are some aspects that are not entirely clear, which I summarize below:

• From the Simple summary and from what is described in the Introduction section (line 65) it is not clear whether antibiotics have already been totally banned in China or the process of banning in this country has been initiated.
• It is also not clear how the 6 animals were selected from each group of the 3 farms. Do those 200 animals belong to each farm or is it a sum of all of them? Where were the animals housed during the study? in the farms?
• The authors' contributions do not conform to the journal's standards.

Author Response

Dear Editor and Reviewers:

Thank you for your letter and for the comments of Reviewers concerning our manuscript entitled “Untargeted metabolomics reveals intestinal pathogenesis and self-repair in rabbits fed an antibiotic-free diet” (ID: animals-1196821). Those comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portions are highlighted in blue. Below please find responses to the comments of reviewers and the modifications to the manuscript.

Reviewer #1

Comment 1: From the Simple summary and from what is described in the Introduction section (line 65) it is not clear whether antibiotics have already been totally banned in China or the process of banning in this country has been initiated.

Response: Thanks very much for the reviewer’s question. I've made some changes (Highlights all amendments (blue)) (lines 65 to 66 on pages 2): China completely banned the use of antibiotics in animal feed in July 2020.

Comment 2: It is also not clear how the 6 animals were selected from each group of the 3 farms. Do those 200 animals belong to each farm or is it a sum of all of them? Where were the animals housed during the study? in the farms?

Response: Thanks very much for the reviewer’s question. I've made some changes. I am very sorry that the inaccurate description of the material method makes the reviewer unclear. "N=3" means not three farms, but three different areas of a breeding shed. There are 3 different areas in a breeding shed, namely "A, B, C", which should be one area the number of rabbit cages is not enough, so we put experimental rabbits in 3 areas to raise, 200 rabbits are all from the same farm. The rabbits of the experimental group and the control group were all randomly selected. During the study period, these rabbits were kept in the farm. The materials and methods have also been revised.

Comment 3: The authors' contributions do not conform to the journal's standards.

Response: Thanks very much for the reviewer’s question. I've made some changes. (Highlights all amendments (blue)) (lines 491 to 493 on pages 16)

Author Contributions: Xianbo Jia, Jie Wang, and Songjia Lai conceived and designed the study; Siqi Xia, Huimei Fan, and Yanhong Li collected data and conducted the research; Tang Tao and Ya Li wrote the paper; Mauricio, A.E. edited the paper.

Reviewer 2 Report

The diet supplied to these animals does not follow any of the current recommendations. I don't know how results have been obtained.

Author Response

Dear Editor and Reviewers:

Thank you for your letter and for the comments of Reviewers concerning our manuscript entitled “Untargeted metabolomics reveals intestinal pathogenesis and self-repair in rabbits fed an antibiotic-free diet” (ID: animals-1196821). Those comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portions are highlighted in blue. Below please find responses to the comments of reviewers and the modifications to the manuscript.

Reviewer #2

Comment 1: The diet supplied to these animals does not follow any of the current recommendations. I don't know how results have been obtained.

Response: I've made some changes (Highlights all amendments (blue)) (lines 89 to 97 on pages 2). The nutritional content of the feed is indeed in accordance with the standard, but it was not expressed in the process of writing the article, for which I feel very sorry: Feed ingredients and additives was prepared according to the French INRA nutrient requirement, and its composition and nutrient content were shown in supporting information Table S2.

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

I have carefully reviewed the work submitted for review and I must say that despite very ambitious results I am not able to make an objective assessment of it.
1. In Europe for 15 years does not apply antibiotics in animal nutrition and there are no problems.
2. The number of results is so enormous that it is difficult for me to verify them.
3. The presentation of the results does not facilitate their verification, on the contrary, it blur the picture even more.
Therefore, it is difficult for me to refer to the content of this manuscript.
For my part, I believe that it should be rejected.

Author Response

Dear Editor and Reviewers:

Thank you for your letter and for the comments of Reviewers concerning our manuscript entitled “Untargeted metabolomics reveals intestinal pathogenesis and self-repair in rabbits fed an antibiotic-free diet” (ID: animals-1147480). Those comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portions are highlighted in yellow. Below please find responses to the comments of Reviewers and the modifications to the manuscript.

Reviewer #1

Comment 1: In Europe for 15 years does not apply antibiotics in animal nutrition and there are no problems.

Response: Thanks very much for the reviewer’s question. Regarding the above issues, I make the following explanations: In recent years, China has only begun to ban the use of antibiotics. After switching to non-antibiotic feeds, the feeding conditions and management levels have not yet reached the production and feeding conditions of European non-resistant feeds. Secondly, the adaptation status of the rabbits has caused deaths from diarrhea. On this basis, this research is to conduct theoretical research to explore its pathogenesis and provide a basis for the research and development of diarrhea drugs. Once again, thank you very much for the reviewer’s comments and suggestions.

Comment 2: The number of results is so enormous that it is difficult for me to verify them.

Response: Thanks very much for the reviewer’s question. The method of this study is based on non-targeted metabolomics, the results are true and effective, and non-targeting is for the purpose of discovery. It is to detect metabolites in biological samples as much as possible, and reflect the total metabolite information to the greatest extent. Metabolite information has discovered some pathogenesis, metabolic pathway differences and other issues, which provide a theoretical basis for subsequent verification tests, and this research group has started follow-up verification tests.

Comment 3: The presentation of the results does not facilitate their verification, on the contrary, it blur the picture even more.

Response: Thanks very much for the reviewer’s question. We have started subsequent experiments according to the results of this paper. The theoretical basis provided by this study for the effectiveness of subsequent experiments is not yet clear, and more experiments are needed to verify it, which we have been doing. However, it is certain that this study provides us with ideas and theoretical basis for subsequent studies. Once again, thank you very much for the reviewer’s comments and suggestions.

Reviewer 2 Report

First of all, I would like to thank you for allowing me to review this article. I have some recommendations to try to improve it:

1. Materials and Methods: the number of animals sacrificed is not described. The description of the groups is also confusing (lines 80-84). How can we obtain 6 animals from 200 farms?
2. The authors should include a table in which the intestinal pathological characteristics are summarized. They only provide two photos in figure 1, but they should give more information: how many animals showed diarrhea? What about the characteristics of this diarrhea, or even different degrees of diarrhea? How many animals showed hyperemia? What about the degree of injury in intestinal wall? and so on.
3. In lines 394-397 they indicated that the liver may be damaged by the change of diet. Is it possible to include data on liver damage? Were liver samples taken from the rabbits?
4. Reference formatting must be improved: no clear rules are followed for all the references.
5. Each number of the references must be separated from the word immediately preceding it.
6. The scientific name of the microorganisms must be in italics.
7. Line 168: Figure 2 should be in brackets.
8. In Figure 2, are you sure that the scale is always the same (100X)? Cannot see blue arrows.

Author Response

please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

GENERAL COMMENT:

There is no information about the diet. As well as no information about the antibiotic used. There are many experiments that explain the effects of a certain nutrient on intestinal health, it is impossible that a work of these characteristics can be understood without this information. There is a lack in the discussion and in the introduction, there are numerous works that deal with the appearance of intestinal diseases with nutritional compositions. They should attach this information prior to publication.

Some other annotations:

• There is no simple simple summary. Should appear
• The contributions of each author should not be put there. There is a special section
• The author's contributions do not follow the typology of animals
• The reference section has a single line.
• The nutritional information or the antibiotic to be used, in what dose it was used, does not appear at all. It is impossible to make a correct discussion of the results if we do not have these data.
• Do you think that six groups per treatment is enough?
• Bold should not be used.
• Complete information is missing from the experimental design.
• There are some abbreviations that are not defined
• A guide to the abbreviations used would help to understand the text
• Tables and graphs should be self-explanatory. Abbreviations that are not correctly explained should not be used (for example Dia / Con, in figure 1)
• There is a word in bold.
• Figures 3, 4 and 6 cannot be read, they should be much larger to understand them.
• Table 1 has "differential" in bold.
• The letter of the bibliography does not match the format of animals.
• Several punctuation marks are missing. Ex (Tab 1, L155…) They should unify the criteria.
• Please, review all format errors.

Author Response

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Author Response File: Author Response.docx

Reviewer 4 Report

In their manuscript “Untargeted metabolomics reveals intestinal pathogenesis and self-repair in rabbits fed an antibiotic-free diet”, authors showed that the pathogenesis of intestinal inflammation using untargeted metabolomics to identify differential metabolites between a group of sick Hyplus rabbit group and healthy Hyplus rabbit group. The study is meaningful and offers suggestion that the antibiotic diet should be changed gradually or alternative antibiotics should be found. There are many aspects that should be improved:

1. The terms such as Fold Change and FC, P-value and P should be consistent in the manuscript.
2. Some words in the manuscript is bold, such as line 165 “rabbit”, line 216 “second”, line 223 “contains” and line 243 “differential”, if they don’t have special meanings, the font should be consistent.
3. For the visible of Figure 2, it’s better to change the color of plotting scale to clearly color.
4. In the results section, “and rectum of Hyplus rabbits from the Dia and Con groups were VIP score >1, FC>1.5 or FC＜0.667 and P < 0.05”. But in the methods section, “Metabolites with variable importance in projection (VIP) > 1, P-value < 0.05, and Fold Change ≥ 2 or FC ≤ 0.5 were considered as differential metabolites”. What’s the difference of the description between result and method?
5. In the line 254, there is mistake in the formula “-log P-value”.
6. The level of English should be improved.
7. The caption of figure in Supplemental Figure S1 should in English.

Author Response

please see the attachment

Author Response File: Author Response.docx