Integrated Transcriptomic and Metabolomic Analysis of the Mechanism of Intramuscular Fat Differences in Wandong Cattle

Dear reviewer:

Thank you for your letter and for the review comments concerning our manuscript entitled “Integrated Transcriptomic and Metabolomic Analysis of the Mechanism of Intramuscular Fat differences in Wandong Cattle” (ID: ijms-3983031). Your comments are all valuable and very helpful for revising and improving our paper. We also thank you for your positive and constructive comments regarding our paper. We have studied your comments carefully and have made corrections which we hope meet with approval. All revised parts have been highlighted in red in the revised manuscript. The main corrections in the paper and the responses to the comments are as follows:

Comments 1: It is recommended to correct the grammar of the title. "Analysis OF the"

Response 1: Dear reviewer, thank you for pointing out this grammatical error in the title. We agree with this comment. Therefore, we have made the necessary revisions. (line 2).

Comments 2: Which characteristic of intramuscular fat is being referred to—profile, distribution, or content? x

Response 2: Agreed. Dear reviewer, thank you for your valuable comment. We can confirm that the characteristic we referred to is the intramuscular fat content. The relevant descriptions in the manuscript have been revised for clarity (line 12).

Comments 3: Correctly name the muscle: *Longissimus dorsi* or *longissimus thoracis*?

Response 3: Agreed. Dear reviewer, thank you for your valuable comment. The text has been corrected to 'Longissimus dorsi' throughout the manuscript (lines 12–13).

Comments 4: It is stated that there are 13 samples, but they are divided into two groups of 4 (8 in total). This information may be confusing in the abstract. In addition, the Methodology section does not explain how the sample size (“n”) for each group was determined.

Response 4: Agreed. Dear reviewer, thank you for your valuable comment. We have provided clearer explanations in the text regarding the process of selecting and grouping the sample. Eight cattle were selected based on similar age and weight. These were then divided into HF (n = 4) and LF (n = 4) groups according to their intramuscular fat content, as measured by the Soxhlet extraction method. This information has been added to both the abstract (lines 14–16) and the methods section (lines 444–447).

Comments 5: L49–52: Too many references are cited for a statement that is already accepted as true by both society and the scientific community. For the statement regarding current changes in consumer preferences for beef quality, you could use articles that directly investigate this point rather than third-party mentions. For example, you may look for studies such as “Consumer perception of marbling and beef quality.”Reference 3 refers to pigs; I recommend using references related to the bovine species.

Response 5: Dear reviewer, thank you for your valuable comment. We agree that since Reference 3 pertains to pig-related research and is redundant, we have removed it (line 53), and cited relevant literature on consumer preferences for beef (line 51).

Comments 6：It is recommended that the authors specify the types of factors being referred to from a disciplinary perspective to make the description clearer.

Response 6: Agreed. Dear reviewer, thank you for your valuable comment. We have changed the first sentence of the introduction to make it clearer what types of 'cellular and metabolic factors' are included. (lines 53-55)

Comments 7：L53. The authors generally mention three factors that affect IMF deposition. However, only the factor related to lipid metabolism homeostasis is developed in the subsequent text. The section on adipose tissue precursor cells is addressed only briefly (it is recommended to strengthen this part). Regarding neuroendocrine regulation, there is no description; the authors should consider whether it is important to develop this aspect.

Response 7: Agreed. Dear reviewer, thank you for your valuable comment. This paper focuses on the regulatory mechanisms of lipid metabolism and provides only a brief overview of fat precursor cells. Neuroendocrine regulation has been removed, as it falls outside the scope of this study. (line 55)

Comments 8: L55. Up to this point, the authors describe fat deposition in terms of “amount or content,” and later refer to it based on its “fatty acid profile.” The authors could improve the wording to make it clearer. It is recommended that they describe these aspects according to their study objectives, as terms such as meat quality, tenderness, and marbling seem to be used interchangeably.

Response 8: Agreed. Dear reviewer, thank you for your valuable comment. We have standardised the terminology throughout the text.

Comments 9: Considering that the objective of this study is related to intramuscular fat, the authors could describe Frezarim’s findings on lipid metabolic pathways in greater detail.

Response 9: Agreed. Dear reviewer, thank you for your valuable comment. We have provided a detailed account of Frezarim's research in lines 57–60.

Comments 10: L80. The authors are encouraged to describe the meat quality of Wandong cattle in comparison with other breeds, such as Angus or Wagyu. This would provide valuable comparative context for the reader. Alternatively, they could refer to other internationally recognized marbling evaluation systems.

Response 10: Agreed. Dear reviewer, thank you for your valuable comment. We have compared the tenderness characteristics of Wandong cattle with those of Angus cattle. (lines 82–84)

Comments 11: The introduction does not address the importance of transcriptomic and metabolomic tools.

Response 11: Agreed. Dear reviewer, thank you for your valuable comment. We have supplemented the importance of transcriptomic and metabolomic tools. (lines 92–104)

Comments 12: L97–98. The sentence is unnecessary, as it is described later and contains a “reference source” error. L100–101. It repeats the same idea regarding “differences in fatty acid profiles between groups.”

Response 12: Agreed. Dear reviewer, thank you for your valuable comment. The sentence contained redundant descriptions regarding intramuscular fat content and fatty acid profiles, and we have removed the redundant portion. (paragraph 2.1)

Comments 13: This error is repeated throughout the entire document.

Response 13: Agreed. Dear reviewer, thank you for your valuable comment. We have thoroughly reviewed the entire manuscript and addressed the issues with figure and table insertion errors.

Comments 14: The authors could include in the Materials and Methods section the mean content and standard deviation for each experimental group (HF and LF) to clearly describe the characteristics of the treatments.

Response 14: Agreed. Dear reviewer, thank you for your valuable comment. We have listed the meant and standard error of the mean for the HF group and LF group (lines 466–467).

Comments 15: I recommend using a more appropriate term than “distribution,” such as “profile” or “characterization.”

Response 15: Agreed. Dear reviewer, thank you for your valuable comment. We have amended 'distribution' to 'profile' in the original text. (line 114)

Comments 16: Is the type of analysis used to identify correlations between variables described in the “Statistical Analysis” section of “Materials and Methods”?

Response 16: Agreed. Dear reviewer, thank you for your valuable comment. This issue arose from my initial unclear wording, which led to ambiguity. I have now revised the original sentence to convey the intended meaning more accurately. (lines 114–116)

Comments 17: Include the common name of the fatty acid alongside its chemical notation, as presented in most parts of the document. Review the entire manuscript to ensure consistency in this aspect.

Response 17: Agreed. Dear reviewer, thank you for your valuable comment. We have added the common name "α-linolenic acid (C18:3n-3)" next to the chemical symbol and will use this format consistently throughout the manuscript. (line 116)

Comments 18: What does “an additional analysis” mean? Is it an unplanned analysis or one suggested by the results?

Response 18: Agreed. Dear reviewer, thank you for your valuable comment. This analysis is a confirmatory comparison that was planned from the outset, and we have replaced 'Further' with 'The' in the text to accurately reflect this. (line 119)

Comments 19: The title of the table should include “fatty acid profile,” as these are the main elements presented in the table.

Response:19: Agreed. Dear reviewer, thank you for your valuable comment. We have added “fatty acid profile” to the Table 1 title. (line 131)

Comments:20: In the Materials and Methods section, the authors should describe what H1, H2, H3, and L1, L2, L3 represent, given that n = 4 per group. If H1, H2, etc., are subsamples, the specific sample handling procedures should be described for all analyses. For each type of analysis performed on the samples, and in each corresponding section, the handling of the samples and/or subsamples should be described, if applicable.

Response:20: Agreed. Dear reviewer, thank you for your valuable comment. We have added the specific definitions of H1, H2, H3, L1, L2 and L3 to the 'Materials and Methods' section, and we have described the sample processing workflow. (lines 469–474)

Comments 21: If each specific sample analysis (fatty acid profile, metabolomic, etc.) has its own statistical analysis applied, these should be described in the Materials and Methods section under “Statistical Analysis.”

Response 21: Agreed. Dear reviewer, thank you for your valuable comment. We have refined the 'Statistical Analysis' section to explicitly list the distinct statistical methods applied to each analysis.(paragraph 4.6, lines 504–510)

Comments 22：It is recommended to carefully review the Discussion section, as several fragments repeat information already presented in the Results, either literally or conceptually. These repetitions make it difficult to clearly distinguish between the description of findings and their interpretation.

In particular, the early part of the discusion reiterates numerical data, group comparisons, and mentions of specific genes or metabolites that have already been described in the "Results section". It is suggested to rephrase these passages to emphasize biological interpretation, comparative analysis with previous studies, and the potential functional relevance of the findings.

Examples:

The sentence “The HF group displayed significantly higher IMF content compared with the LF group” repeats text already included in the Results (Section 2.1). In the Discussion, it could instead be rephrased as an interpretation, for example: “The greater IMF content observed in HF cattle may be explained by enhanced lipid deposition pathways.”

Similarly, “Genes such as FABP4, SCD, and FASN were significantly upregulated in the HF group” reiterates transcriptomic findings (Section 2.2). Rather than restating the result, it would be preferable to discuss how the upregulation of these genes could promote fatty acid synthesis or lipid storage.

Response 22: Agreed. Dear reviewer, thank you for your valuable comment. We have revised the Discussion section by removing redundant content and focusing on the biological interpretation of the findings and their significance. (paragraph 3)

Comments 23: L308–313 This section is not part of the discussion; it describes background information and results that have already been presented in the “Results” section.

Response 23: Agreed. Dear reviewer, thank you for your valuable comment. We have removed the duplicate content on lines 308-313.

Comments 24: L313–319 The authors should discuss the results obtained rather than merely repeating them. Factors that may explain the variations in results between groups—such as age, sex, and diet composition—are mentioned. These factors should be further developed and discussed (and the characteristics of the Wandong cattle should also be included in the Methodology section).

Response 24: Agreed. Dear reviewer, thank you for your valuable comment. We have revised the Discussion section to focus on interpreting the results and exploring potential mechanisms. The characteristics of Wandong cattle have been added to the Methodology section (lines 444–445). Since all experimental animals were of similar age, sex, and raised under identical conditions, we have emphasized that the observed variations are more likely attributable to intrinsic molecular regulation (lines 326–330).

Comments 25:

It is important to describe the characteristics of the animals, such as age, weight, and whether they come from the same group. Environmental factors such as diet and the physiological maturity of the animals influence intramuscular fat deposition. The collection of samples with different intramuscular fat contents should result from intrinsic factors and not, for example, from differences in age or diet when animals come from different production units.

This distinction is crucial to clarify the hypothesis—whether the differences observed in the analyses of the samples are due to intrinsic factors of the animals (genetic) or simply to intramuscular fat content influenced by diet (extrinsic factors).

Response 25: Agreed. Dear reviewer, thank you for your valuable comment. We have added supplementary information regarding the animal characteristics in the “Materials and Methods” section. (lines 441-445).

Comments 26: Clarify whether the animals were purchased and immediately slaughtered, or if any handling or adaptation period was carried out—if so, specify the duration (in days) and the conditions.

Response 26: Agreed. Dear reviewer, thank you for your valuable comment. Our laboratory animals were fasted for 24 hours after purchase, followed by sampling (line 447).

Comments 27: Describe the sample collection procedure in detail—specify which portion of the *longissimus dorsi* was used (for example, *longissimus thoracis*), which part of the *L. thoracis* was sampled, and from which side of the carcass. Provide a more detailed description.

Response 27: Agreed. Dear reviewer, thank you for your valuable comment. We have revised the Methods section to include the precise anatomical location of muscle sampling and refined the description of the sample processing procedure. (lines 449–455)

Comments 28: There should be a reference procedure describing the sample characteristics, as the term “smaller pieces” is subjective. For instance, how many grams in total are required for each of the subsequent analyses?

Response 28: Agreed. Dear reviewer, thank you for your valuable comment. We have explicitly labelled the subjective description 'smaller fragments' as '10 g per sample' in the text for subsequent analysis. (line 452)

Comments 29: Specify which international organization or whether it is a national standard that GB 5009.6-2016 belongs to.

Response 29: Agreed. Dear reviewer, thank you for your valuable comment. We have explicitly added the following to the text: GB 5009.6-2016 and GB 5009.168-2016 are national food safety standards in China and are widely adopted as authoritative specifications for food safety and testing. (lines 457–458 and 460)

Comments 30: In general, the Materials and Methods section should clearly describe the sampling process and how the samples were used in each analysis. This information is not provided in each subsection corresponding to the different analyses performed on the samples.

For example, all data were obtained from a sample size of n = 4 per group; however, samples were collected from 13 animals in total. In L459, the authors state that the samples were classified into two groups, HF and LF, but the distribution of these groups and the criteria used to select n = 4 per group for the analyses are not clearly described.

The authors should specify, for each analysis performed, how the samples were handled, how many subsamples or replicates were made, and how the mean value for each n was obtained.

Response 30: Agreed. Dear reviewer, thank you for your valuable comment. We have supplemented the sample processing workflow for each analysis in the “Materials and Methods” section and explicitly stated: experiments selected 8 cattle with similar age and weight from a total of 13 animals, dividing them into HF and LF groups based on intramuscular fat content (n=4 per group). We have also refined the sample processing methods, number of replicates, and n-value standards for each analysis. (paragraph 4, line 439)

Comments 31：The conclusion is too brief and does not reflect the breadth of the study’s findings. It is limited to mentioning a few genes and signaling pathways but fails to synthesize the main experimental results or discuss their relevance and applicability in a productive or scientific context.

It does not integrate the three levels analyzed (IMF content, transcriptomics, and metabolomics).

It does not address the importance of the findings in terms of genetic improvement, meat quality, or potential practical applications.

It does not provide a conclusive perspective on how the results contribute to the overall understanding of the molecular control of IMF in local Wandong cattle.

Response 31: Agreed. Dear reviewer, thank you for your valuable comment. We have revised the conclusion to better synthesize our multi-omics findings and highlight their scientific and practical implications. (lines 512-521)

Comments 32：Meat quality encompasses multiple generally desirable characteristics. I recommend that the authors specify which particular characteristic was analyzed such as intramuscular fat content or fatty acid profile and discuss the corresponding results accordingly.

Response 32: Agreed. Dear reviewer, thank you for your valuable comment. We have explicitly stated in the conclusion that the meat characteristics analyzed in this study are intramuscular fat content and fatty acid composition.