Maternal Tributyrin Supplementation During the Perinatal Period Is Associated with Improved Ewe Milk Quality and Lamb Growth Performance, Immunity, and Antioxidant Status

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear editor,

I have read the manuscript with attention. In general, I found it well written, interesting and informative. However, it requires some changes prior to a potential publication in the journal. I ammended those suggestion in the attached file to this report.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English require minor changes.

Author Response

We sincerely thank you for your valuable comments and suggestions, which have been greatly beneficial to improving our manuscript. A detailed, point-by-point response to each comment can be found in the attached documents.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Comments to Authors

General Comments:

The authors address a practical research question and present data of considerable interest to the field. However, several concerns require substantive revision before the manuscript can be considered suitable for publication.

Specific Comments

Comment: The current title contains grammatical errors and appears to overstate the findings of the study. I recommend revising the title to more accurately reflect the scope and conclusions of the research. Please replace existing title with suggested one:

"Maternal Tributyrin Supplementation During the Perinatal Period Improves Ewe Milk Quality and Enhances Lamb Growth Performance, Immunity, and Antioxidant Status".

Comment 1 (Lines 93-95): The randomization procedure is not adequately described. Please specify the method used to randomize ewes into groups (e.g., simple randomization, stratified randomization) to ensure reproducibility and assess potential selection bias.

Comment 2 (Lines 103-107): Study design lacks a proper positive control group (100% NRC without tributyrin). The observed effects cannot be attributed solely to tributyrin since the NEB group was intentionally underfed. Please acknowledge this limitation.

Comment 3 (Lines 110-111): The sample size of n=5 lambs per group for slaughter analysis is relatively small. Please provide a power analysis or justification for this sample size, and acknowledge this limitation in the discussion.

Comment 4 (Table 1, Lines 112-115): The chemical composition values appear to represent only one diet formulation, yet two different feeding levels were used. Please clarify whether these values apply to both groups or provide separate nutritional profiles for NEB and TB diets.

Comment 5 (Lines 116-127): The apparent digestibility methods describe measurements in "five lambs from each group" using metabolic cages, yet Table 5 header (Line 215) refers to "Hu sheep." Please clarify whether digestibility was measured in ewes or lambs, as this is confusing.

Comment 6 (Lines 223-224): There is a significant error in terminology: "alkaline phosphatase (AST), alkaline phosphatase (ALT), alkaline phosphatase (ALP)." AST is aspartate aminotransferase and ALT is alanine aminotransferase—these are not alkaline phosphatase. Please correct this error.

Comment 7 (Lines 129-130): Blood collection is stated as "7:00 on days 7 and 42 (before feeding)." Please specify the fasting duration prior to blood collection and clarify if this timing was consistent across all animals.

Comment 10 (Lines 277-279): The statement regarding differences between your findings and previous studies mentions "maternal supplementation rather than direct feeding." Please elaborate on the biological mechanisms by which tributyrin metabolites may transfer through milk to offspring, as this is central to your hypothesis.

Comment 11 (Figure 1, Lines 242-247): The y-axis label "Relative gene expression" lacks units or normalization method description. Please indicate that expression was normalized to ACTB and specify if data represent fold-change relative to the control group. Specify normalization method and whether data represent fold-change relative to control.

Comment 12 (Line 330): Grammatical error: "first examine" should be "first to examine."

Comment 13 (Lines 51, 71): Minor grammatical corrections needed: "nutrients deficiency" should be "nutrient deficiency" (Line 51); "balances input microbial communities" is unclear and should be revised (Line 71).

Comment 14 (Tables 6-8): Please report exact p-values rather than only indicating significance thresholds (e.g., report p=0.037 rather than just P<0.05) for transparency, which you have done in some tables but not consistently.

Comment 15 (Lines 329-342): The conclusions make strong claims about this being "the first" study of its kind. Please verify this claim with a more thorough literature search, and consider tempering the language to acknowledge study limitations including small sample size and the specific breed (Hu sheep) which may limit generalizability.

Comment 16 Overstated novelty claims:

• "first examine tributyrin supplementation during gestation" (Line 330)
• "first to systematically reveal" (Lines 336-337)

Comment 17 Conclusions exceed the data: The conclusion states tributyrin "effectively alleviates nutritional stress caused by NEB" (Lines 334-335), but the study cannot distinguish tributyrin effects from the effects of simply providing adequate nutrition.

Comment 18: The conclusion should be rewritten to:

• Summarize key findings objectively
• Acknowledge that effects cannot be attributed solely to tributyrin due to study design
• State limitations clearly
• Recommend future studies with proper control groups
• Provide cautious practical implications

Comment 19 Logical gap in immune discussion (Lines 301-314): The jump from discussing fetal inflammatory response syndrome (Line 309-310) to concluding that tributyrin helps establish the "initial immune system" (Line 311-312) is not well supported by the data presented.

 

Comment 20 Superficial comparison with previous studies (Lines 272-279): When discussing conflicting results from other studies, the explanation ("differences in duration" and "maternal vs. direct supplementation") lacks depth. What specific biological mechanisms explain these differences?

Comment 21: Incomplete mechanistic explanation (Lines 277-284): The authors claim maternal supplementation transfers benefits through milk, but they do not:

• Measure tributyrin or butyrate concentrations in milk
• Explain how tributyrin metabolites reach offspring
• Discuss whether butyric acid survives digestion and reaches lamb tissues

Author Response

We sincerely thank you for your valuable comments and suggestions, which have been greatly beneficial to improving our manuscript. A detailed, point-by-point response to each comment can be found in the attached documents.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This study focuses on the effects of tributyrin supplementation in periparturient ewes with negative energy balance (NEB) on the growth, immunity, and antioxidant status of their lambs. The research topic is closely linked to practical issues in sheep breeding production, holding significant practical guiding significance and theoretical value. The study design follows a clear logical framework: through rigorous group-controlled trials, systematic sample collection, and multi-dimensional index detection, it systematically explores the mechanism of action of tributyrin, filling the research gap in this field regarding sheep. The overall research framework is complete, with relatively sufficient data support.

Revision Suggestions

1, Clarification of Feed Nutrient Composition

In Table 1, the "late pregnancy feed" only lists the overall values of some nutrients (e.g., metabolizable energy, crude protein) for the NEB group and TB group, without specifying whether these nutrients differ between the two groups due to tributyrin addition. It is recommended to supplement the specific nutrient values of late pregnancy and lactation feeds for each group, clarify whether tributyrin addition affects the basic nutritional level of the feed, and ensure that tributyrin is the only experimental variable.

2, Details on Milk Collection and Feeding

The text mentions that "ewe milk was collected at 06:00, 10:00, 14:00, 18:00, and 22:00 daily and then fed to the corresponding lambs" but does not specify the milk preservation method after collection or whether the feeding dosage was adjusted based on lamb body weight or age. It is recommended to supplement information on milk preservation conditions (e.g., temperature, storage time), the basis for determining the daily milk feeding amount for lambs, and the adjustment plan to avoid inaccuracies in lamb growth data caused by improper feeding dosages.

3, Supplementary Information on Gene Expression Detection

For the quantitative real-time PCR section, only β-actin (ACTB) is mentioned as the reference gene, but the results of reference gene stability verification (e.g., GeNorm or NormFinder analysis) are not provided. It is recommended to supplement the stability verification data of the reference gene to ensure the reliability of gene expression quantification results. Additionally, the labeling order of the forward (F) and reverse (R) primers for the GSH-Px gene in Table 2 is inconsistent and needs to be corrected to clarify the corresponding relationship of primer sequences.

4, Details on Apparent Digestibility Detection

The text states that "5 lambs were individually housed in metabolic cages, and feces and urine were collected for 5 days" but does not specify the isolation effectiveness of the metabolic cages (e.g., whether cross-contamination between feces and urine was avoided) or whether there was an acclimation period before sampling. It is recommended to supplement details on the use of metabolic cages (e.g., isolation design) and the acclimation time of lambs to the cages, and clarify the specific operation procedures for feces and urine sampling (e.g., whether samples were collected at fixed times daily, whether sampling tools were sterile) to ensure the accuracy of digestibility data.

5, Standardization of Data Tables

The column of "milk composition" in Table 3 is confusingly presented, with unclear correspondence between the values of lactose, protein, fat and the experimental groups. It is recommended to reformat the table to clearly show the specific values of each nutrient in the NEB group and TB group.

The unit labels for "crude protein", "crude ash", and "gross energy" in Table 5 are missing and need to be supplemented (e.g., crude protein as "%", gross energy as "MJ/kg").

Group labels should be consistent across all tables. In Table 3, "TBG=0.5% tributyrin supplementation group" is recommended to be unified as "TB=0.5% tributyrin supplementation group", consistent with the abstract and other tables.

6, In serum biochemical indices, the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in the L-NEB group were significantly higher than those in the L-TB group, suggesting that NEB may cause liver function damage in lambs. It is recommended to supplement the interpretation of this biological significance in the result analysis and correlate it with the protective effect of tributyrin on lamb liver function.

7, For antioxidant indices, the liver GSH-Px activity in the L-TB group showed an increasing trend (P=0.063), approaching a significant difference level. It is recommended to combine the gene expression results (significantly increased liver GSH-Px mRNA levels) to analyze the correlation between enzyme activity and gene expression, and explain the potential post-transcriptional regulatory mechanisms.

8, The current discussion section explains the effects of tributyrin on inflammatory factors and antioxidant enzymes based on existing studies but does not mention the specific mechanisms by which it improves ewe milk quality (e.g., how tributyrin regulates milk fat and milk protein synthesis in mammary epithelial cells), as well as the metabolites and transmission efficiency of tributyrin in milk. It is recommended to supplement relevant mechanistic discussions, incorporating the regulatory role of short-chain fatty acids in mammary gland development and nutrient synthesis to enhance the theoretical depth of the study.

9, Explanation of Research Limitations

This study only set one tributyrin supplementation dosage (0.5%) and did not explore the effects of different concentrations, making it impossible to determine the optimal addition level. It is recommended to mention this limitation in the discussion and provide a reference for future research directions (e.g., dose-effect trials). Additionally, the study did not detect changes in ewe intestinal flora and metabolites; it can be inferred that tributyrin may indirectly affect milk quality by regulating ewe intestinal health, and it is recommended to supplement the discussion on this potential action pathway.

10, Comparison with Existing Studies

The discussion mentions differences between the study results and those on dairy cows and piglets but does not clarify the specific reasons for such differences (e.g., animal species, physiological stages, supplementation methods). It is recommended to further refine the comparative analysis, clarify the mechanism underlying the differential effects of tributyrin supplementation in periparturient ewes versus direct supplementation in young animals, and enhance the interpretation of the universality and specificity of the study results.

11, Correction of Textual Errors

In the abstract, "serum IL-1β concentra- November concentration" is an obvious typo; "November" should be deleted, and the correct expression is "serum IL-1β concentration".

In the discussion, "IL- -January and IL-6 are endogenous sources of heat" should be corrected to "IL-1β and IL-6 are endogenous sources of heat".

The legend of Figure 1 is incomplete, with missing content after "no letters or the"; it needs to be supplemented to clarify the meaning of data labeling.

 

Author Response

We sincerely thank you for your valuable comments and suggestions, which have been greatly beneficial to improving our manuscript. A detailed, point-by-point response to each comment can be found in the attached documents.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all of my comments thoroughly and satisfactorily. I have no further concerns.

Author Response

We sincerely thank the Reviewer for the positive feedback and for confirming that all comments have been adequately addressed. We appreciate the Reviewer’s careful evaluation and valuable time.

Reviewer 3 Report

Comments and Suggestions for Authors

In light of the reviewers' comments, the authors have conducted comprehensive revisions, resulting in substantial improvements to the manuscript. I therefore recommend its acceptance and publication.

Author Response

We sincerely thank the Reviewer for the positive feedback and for confirming that all comments have been adequately addressed. We appreciate the Reviewer’s careful evaluation and valuable time.