Transcriptomic and Metabolomic Insight into the Roles of α-Lipoic Acid in the Antioxidant Mechanisms of Sheep

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General comments.

Since the authors have previously published data, they have written a confusing and unclear manuscript. The Abstract should be rewritten since it contains combined information from a previously published investigation and the present study. The introduction should be improved. In its current form, the authors did not expose the relevance of the current study. There is not a clear hypothesis. The materials and methods are confused and incomplete. The treatments are unclear. The discussion is insufficient.

 

Specific comments:

Lines 17 to 20. These are data from another study.

Line 26. Define the abbreviation “PPP”.

Lines 46 to 54. There are already known mechanisms by which a-LA exerts its antioxidant and immunological effect, so the authors should say what is unknown about it or why transcriptomic and metabolomic studies are necessary.

Materials and methods

Lines 68 to 82. The authors used samples obtained from a previous study. This should be explicitly mentioned in materials and methods. Not all treatments from the previous study should be mentioned, but only from which treatments the samples for this study were obtained, otherwise the information becomes confusing. What were the treatments for this specific study?

Lines 78 to 82. What was the criterion for choosing only six sheep? How many sheep were killed for each treatment? Why do the authors mention obtaining blood samples? What were the serums used for in this study? Unnecessary information should be removed or what they were used for should be clarified.

Lines 79 to 80. The description of the rumen sample should be expanded. How did you ensure that the sample was homogeneous? How much rumen tissue was collected? Were they fragments of the entire rumen or just the epithelial layers? What portion of the rumen was sampled?

Lines 87 to 93. Please provide more information on the methods and equipment (including brand and model) used to determine the antioxidant and anti-inflammatory activity of rumen tissue.

Lines 115 to 136. It is not clear which variables were determined. Please explicitly state which variables were determined.

Lines 146 to 150. What was the statistical design used to evaluate the treatments? Describe all statistical analyses and comparisons performed.

Lines 163 to 166. Delete. It is NOT part of the results of this study. In any case, send it to the introduction.

Lines 168. Delete the text ‘We investigated the effect of α-LA on gene expression in rumen epithelial tissue’.

Results

Line 170. Differences between treatments (α-LA vs CON) are mentioned in the text, however, this information is not shown in the Figures and the text of the Results section.

Figures are small and difficult to read.

 

230-272. The discussion is vague at the beginning. It contains only general information that does not attempt to explain the findings of the current study, their comparison with other similar published studies, or their possible implications. The discussion seems more like a literature review than a discussion.

Comments on the Quality of English Language

A moderate revision of the English language may benefit the understanding of the manuscript.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1.       Abstract written supportively I have no comments

2.       Introduction written in very general terms, no purpose of the study, should add some information on the role of acid , α-LA, synthesis of

3.       Material and methods ;No description of animal nutrition given only acid doses of 0 (CTL), 300 (LA300), 450 (LA450), 600 (LA600), and 750 (LA750) , no what feed was used, what were the maintenance conditions, the system of maintenance ( pasture or in the building), what was the temperature in the buildings

4.       Results described correctly

5.       The discussion needs to be revised, referring to the results obtained.

6.       The conclusion should include what parctical significance the research has, 

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript discusses the effects of the dietary supplementation of alpha lipoic acid in sheep diets on their antioxidant mechanism This subject is of interest, but the manuscript requires several revisions. Also, I believe that the percentage of plagiarism is too high.

Abstract

In line 12, the authors should format the second phrase according to the entire manuscript.

In lines 12-13, the authors should define that the supplement was added to the sheep diet, and the doses used.

In lines 15-16, please correct with “…… the role of α-LA in antioxidant and inflammatory status”

Introduction

Line 33: The authors must correct the comma with a period.

Line 44, please correct the period with a comma.

Line 45, please avoid using the word “and” doubled in the same phrase consecutively.

Please clearly present the aim of the study in this chapter.

In this chapter, the authors should add several references that present the effects of alpha lipoic acid in ruminants, in addition to their previous study.

Materials and methods

2.1 Animal experiment and sample collection

Line 68: This chapter has to present the average of the Duhu F1 hybrid age.

The authors should present the ingredients of the diet. Also, it has to be presented the intakes of every experimental diet.

The authors also have to present the α-LA supplementation methods.

Please present the details of the supplement such as the administration methods, and form.

The authors should detail the slaughtered method.

Please specify how the supplementation levels of the α-LA were decided.

2.2. Analysis of antioxidant and anti-inflammatory activities of rumen epithelial tissue

The authors should add information on the determinations presented in this subchapter, and also present the full name of the antioxidant parameters determined, before using the abbreviation.

2.3. Transcriptome sequencing of rumen epithelial tissue

Line 97, the authors should add detail for the Qubit.

2.5. Metabolite identification and analysis of rumen epithelial tissue

The authors should present the manufacturer and its country, for the UPLC.

2.6. Statistical analysis

For the heatmap, the authors have to present the color codes for the correlations.

Results

The study did not discuss the analysis of the antioxidant status. However, the method for determining the antioxidant status is detailed in the Materials and Methods section. Please also mention in the Materials and Methods chapter that the determinations and discussions of these parameters were previously presented, and that the current data are being used only for correlation purposes.

For Figure 1, please clarify the heatmap to improve readability.

Discussions

Lines 230-233, please add references.

In this chapter, the authors should add references to studies that used α-LA in ruminants or other animals, detailing the doses used and the results obtained.

The study involved five different diets, please detail which was the most effective.

Conclusions

In this chapter has to be added the future perspectives and the limitations of the study.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed most of the requested corrections successfully. Only two additional comments are requested to be addressed.

Specific comments.

Line 157: Please clarify the meaning of the abbreviation "DGEs."

 

In the Results section and Figures 1 and 3, please include the probability values (P-values) when discussing the differences between treatments (α-LA vs. CON). Additionally, ensure that these P-values are also reflected in Figures 1 and 3.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Accepts amendments

Author Response

Thank you very much for your reiview

Reviewer 3 Report

Comments and Suggestions for Authors

I have reviewed the revised manuscript and appreciate the authors’ efforts to address my suggestions. However, there are still several information that need to be added.

Firstly, please provide the ingredients of the diets. If these were included in previous work, it would be helpful to indicate whether the ratios are balanced in terms of energy and nitrogen intake.

Additionally, please specify the procedure for incorporating α-LA into the animals’ diets. Since the effects of dietary α-LA are discussed, it is essential to present the intake levels of the diets as well.

Author Response

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Author Response File: Author Response.pdf