Establishment of Flavonoid Fingerprint of TMR Diet and Optimization Factor Analysis Strategy and In Vitro Fermentation Parameters Based on Spectrum–Effect Relationship

Round 1

Reviewer 1 Report

## Comments on abstract, and title

- The title is relevant to the aim of the study. This study has a good idea to study conducted to identify nutricines in the total mixed ration (TMR) and provides insights into feed quality and their impact on dairy cow health.

- the aim of the study is very clear “ Establish a chemical fingerprint analysis of TMR diets for compound prediction of spectrum-structure-activity”

## Comments on the introduction

This study introduces the importance of nutricines, the nutritional active substances in feed, which play vital roles in enhancing immune function, antioxidant activity, and feed efficiency in dairy cows. So, the topic of this paper is clear. Moreover, the research question sightly appears.

But I have some comments

-       In lines 44 and 45: the authors took about: Flavonoids are classified under polyphenolic compounds as they possess A and C rings of benzo-1-pyran-4-quinone and a B ring.   

My comment: the authors have not described all types of flavonoids.

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## Comments on methodology

- the methods used in this study were valid and reliable

- I am satisfied that to use of a good number of TMR diets (20 diets).

But I have some comments on this point (its advice):

-       I suggested that: characterizes diet, and experimental design through a schematic form.

-       the authors have not described the diet which contains in a table.

## Comments on data results and discussion

The paper is generally well-written and structured.

-       Figure 1. (a) HPLC chromatogram of S19 solution. (b) HPLC chromatogram of seven standards. 

-       Figure 2. Fingerprints of TMR from different cattle farms. Need more resolution

Comments on  In vitro rumen fermentation results

-       In Lines 166, 167.  Data were generally shown that the dry matter disappearance rates of S17 and S19 are higher than those of other samples, While in lines 181 and 182: In general, the VFA content of S2, S4, S7, and S18 was relatively low, while the content of S11 was relatively high.

My comment: if comparing the dry matter disappears and acetate or VFA production should diet S11 have a high dry matter disappearance rate? So, I advise the authors to explain and revise this point

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-       ## Comments on conclusions

- the conclusions answer the aims of the study: The authors conducted a qualitative analysis of 7 flavonoids in 20 TMR diets of dairy cows, namely: p-coumaric acid, sinapic acid, quercetin, tricin, apigenin, luteolin, and diosmetin.

- I think scientists have opportunities to inform future research and more research is needed to confirm others' nutricines.

- I suggest updating some references.

Author Response

Thank you very much for your comments. I will try my best to revise the manuscript to achieve the best. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

I have reviewed the manuscript, and here are the comments:-

English needs to improve; there are some sentences and parts of the manuscript that are difficult to understand

Many abbreviations in the manuscript need to be cleared, and add more results in the abstract.

Please arrange all keywords in alphabetical order

Provide the origin and model of all devices.

Table 9. Putative structure-active compounds based to the cluster, please add the source of structure-active compounds… add a citation

To improve the quality of the paper, update the reference list by adding 2022 and 2023 references.

Please follow the authors' instructions on how they write the reference in the list. Why are you capitalizing the first letter of every word? Please see the journal style. For references about textbooks, please add the page numbers of the textbook. Also, please add the city of the publisher.

Please make all tables self-explanatory and do not use abbreviations in the table footnote or legend.

Enhance the resolution of all figures.

The conclusion shouldn’t contain results, check and rewrite this part.

English needs to improve; there are some sentences and parts of the manuscript that are difficult to understand.

Author Response

Thank you very much for your comments. I will try my best to revise the manuscript to achieve the best. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

My major concern with this paper is the lack of inclusion of materials and methods. The authors fail to adequately describe the methodology used in the present experiment or statistical analysis used to obtain the results. Therefore, the results section is difficult to comprehend or evaluate, along with the discussion.

Minor editing required.

Author Response

Thank you very much for your comments. I will try my best to revise the manuscript to achieve the best. Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have carefully processed all comments. The quality of the manuscript has increased significantly. I have no further comments.

Minor editing of the English language required