Intestinal Development in Wuzhishan Pigs at Different Growth Phases: Insights from Microbiome and Metabolomics

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses an interesting question using intestinal morphology, 16S rRNA profiling, and untargeted metabolomics across developmental stages in Wuzhishan pigs. Several issues substantially limit scientific reliability and interpretation. Author should clarify and justify the stage definition because each stage pools multiple ages, and key metadata (sex, litter, batch) and their control are not adequately reported, creating major confounding. Please re-check and correct the diet tables, as the reported crude fat values appear implausible and diet is a dominant driver of microbiome and metabolome changes. Please report contamination controls and consider ASV-based analysis rather than 97% OTUs, and avoid labeling taxa as harmful at genus level. Author should strengthen metabolomics reporting by providing identification confidence levels, QC filtering metrics, and verification of biologically questionable compounds, and interpret PLS-DA and microbe–metabolite correlations cautiously with FDR correction. Finally, please tone down causal language and feeding recommendations, as the cross-sectional observational design does not support mechanistic conclusions.

Comments for author File: Comments.pdf

Author Response

Dear Editor and Reviewers,

On behalf of the authors of the submitted manuscript “Evaluating Different Stages of Intestinal Development in Wuzhishan Pigs Through Microbiome and Metabolomic Analysis”, we sincerely thank you for your valuable reviews and suggestions. These comments are very helpful. We have carefully studied the comments and made corresponding revisions, which we hope will meet with your approval.The main revisions in the paper (changes highlighted in yellow) and the responses to the reviewer’s comments are as follows:

Responses to the reviewer’s comments:

 

Responds to Reviewer 1:

• Author should reduce causal conclusions because the design is cross-sectional and heavily confounded by diet and age pooling. 

AU: We thank the reviewer for the valuable suggestion. We have revised the causal conclusions throughout the manuscript to avoid overinterpretation, in consideration of the cross-sectional study design.

 

• Please remove marketing-style wording (high-quality miniature pig breed) and replace with objective traits relevant to the study.

AU: We thank the reviewer for the valuable suggestion.We have removed the marketing-style description “high-quality miniature pig breed” from the abstract and the entire manuscript, and replaced it with: “Wuzhishan pigs are a typical Chinese indigenous miniature pig breed”, and highlighted in yellow in L34-35.

 

• Author should define what different developmental stages means (exact ages) already in the abstract.

AU: We appreciate the constructive comment.We have clearly defined the exact ages of the four developmental stages in the abstract:pre-weaning stage (7 and 14 days old), weaning stage (35, 38 and 45 days old), fattening stage (70 and 100 days old), and maturity stage (180 and 240 days old), and highlighted in yellow in L37-39.

• Please avoid calling genera harmful bacteria at genus level (Streptococcus/Romboutsia/Terrisporobacter include diverse species), 16S genus-level does not prove pathogenicity.

AU: We thank the reviewer for the valuable suggestion. We have revised “harmful bacteria” to “potentially opportunistic pathogens” in the abstract, and highlighted in yellow in L44-45.

 

• Author should be careful claiming lipid metabolism was significantly lower because the study measured luminal metabolites, not host metabolic flux.

AU: We thank the reviewer for the valuable suggestion. We have revised “lipid metabolism” to “lipid metabolite levels” to accurately reflect that luminal metabolites rather than host metabolic flux were measured, and highlighted in yellow in L41-46.

 

• Author should avoid implying Lactobacillus causes muscle development; the study has no intervention.

AU: We thank the reviewer for the valuable suggestion. We have adjusted the sentence structure to avoid implying a causal effect of Lactobacillus on muscle development–related metabolites, and highlighted in yellow in L46-49.

 

• Author should reduce excessive descriptive breed background and focus more on the scientific gap and hypothesis. 8) Please clarify what is meant by nutritional needs because no nutritional requirement experiment is performed.

AU: We thank the reviewer for the valuable suggestions. We have reduced the excessive descriptive background of the breed and supplemented the scientific gap. In addition, we have revised “nutritional needs” to “physiological characteristics” to avoid misunderstanding, as no nutritional experiment was performed in this study. These revisions are located in lines 56-68.

 

• Author should avoid over-generalized microbiome function claims without linking to pig-specific evidence.

AU: We thank the reviewer for the valuable suggestion. We have revised the over-generalized statements regarding microbiome functions, and highlighted the revisions in yellow in L87-95.

 

• Author should avoid mechanistic claims like (secondary cholic acids derived from Lactobacillus…) unless bile acid transformation was measured directly.

AU: We thank the reviewer for the valuable suggestion. We have revised the mechanistic statements regarding microbial metabolites and bile acids. The revisions are highlighted in yellow in L102-106.

 

• Author should clarify sample size and statistical model, weights are compared across stages but each stage contains multiple ages. Please explain why fattening vs weaning shows no significant weight difference if the age gap is large; this suggests major within-stage variability or statistical issues.

AU: We thank the reviewer for the valuable comments. We have clarified the sample size of each stage and the statistical model used for body weight comparison. We have also added an explanation for the non-significant difference between the weaning and fattening stages, which may be due to high individual variation and slow weight gain during these periods. The revisions are highlighted in yellow in L250-253 and L257-258.

 

• Please specify whether histology scoring/measurement was blinded. 13) Author should address contradictory interpretation, VH decreases at weaning but VW increases; discuss whether villus edema or orientation bias could affect VW.

AU: We thank the reviewer for the valuable comments. We have clearly stated that all histological measurements were conducted in a blinded manner. We have addressed the seemingly contradictory finding that villus height was decreased while villus width was increased during the weaning period in the discussion section. The revisions are highlighted in yellow in L256-266 and L587-592.

 

• Author should avoid claiming enzyme activity directly reflects digestive capacity without linking to growth efficiency or digestibility.

AU: We thank the reviewer for the valuable comment. We have revised the description of intestinal enzyme activity and avoided overstating its direct reflection of digestive capacity. The revisions are highlighted in yellow in L299-301.

 

• Author should correct OUTs to OTUs.

AU: We thank the reviewer for the valuable comment. We have corrected all OUTs to OTUs throughout the text. The revisions are highlighted in yellow in L333-340.

 

• Author should remove speculative explanations (roughage tolerance, sensitivity) unless diet composition and fiber intake were measured and compared.

AU: We thank the reviewer for the valuable comment. We have removed the speculative explanations regarding roughage tolerance and dietary sensitivity, as diet composition and fiber intake were not measured in the present study. The revisions are highlighted in yellow in L352-356.

 

• Author should avoid concluding Actinomycetes enrichment at infant stage without noting whether contamination controls were used.

AU: We thank the reviewer for the valuable comment. We have revised the description of Actinomycetes abundance. The revisions are highlighted in yellow in L375-378.

 

• Please fix spelling inconsistency: Terrrisporobacter vs Terrisporobacter vs Terrisporobacterium.

AU: We thank the reviewer for the valuable comment. We have corrected the spelling inconsistency of Terrisporobacter. The revisions are highlighted in yellow in L386.

 

• Please provide actual QC metrics (RSD %, missingness, drift correction) not only qualitative statements.

AU: We thank the reviewer for the valuable comment. We have supplemented the actual QC metrics including RSD %, missingness, and drift correction in the results section. The revisions are highlighted in yellow in L409-413.

 

• Please shorten extremely long sentences and avoid embedding interpretation into the results section, move mechanistic claims to Discussion.

AU: We thank the reviewer for the valuable comment. We have shortened all extremely long sentences and completely removed interpretative and mechanistic claims from the Results section, which will be integrated into the Discussion section in subsequent revisions. The revisions are highlighted in yellow in L426-455 and L731-761.

 

• Author should specify enrichment method, background set, and multiple-testing correction for KEGG. Author should justify dopaminergic synaptic pathway enrichment, this often appears in metabolomics due to annotation overlap and should not be over-interpreted.

AU: We thank the reviewer for the comment. We have specified the KEGG enrichment method (hypergeometric test), background set (all annotated metabolites), and multiple-testing correction (Benjamini–Hochberg) in the revised manuscript. The dopaminergic synaptic pathway has been removed from the Results section. The revisions are highlighted in yellow in L469-474 and L479-481.

 

• Author should report the number of correlations tested and apply FDR correction, otherwise false positives are expected. 23) Please reconsider interpreting Fusobacteria as beneficial in lipid metabolism, Fusobacterium is often linked with inflammation in many hosts.

AU: We thank the reviewer for the valuable comments. FDR correction was applied for multiple correlation tests to control false positives. We have added a cautious statement to clarify that the association between Fusobacteria and lipid metabolites does not confirm a beneficial role in lipid metabolism, and its exact function requires further investigation. The revisions are highlighted in yellow in L493 and L504-506.

 

• Author should be cautious interpreting bile acid pathways, 16S genus-level cannot confirm bile acid transformation capability.

AU: We thank the reviewer for the comment. We have added a cautious statement indicating that genus-level 16S rRNA sequencing cannot confirm bile acid transformation capacity, which requires further experimental verification. The revisions are highlighted in yellow in L512-514.

 

• Author should avoid comparing final weight to commercial breeds without stating that Wuzhishan pigs are miniature, the comparison is expected and not informative.

AU: We thank the reviewer for the comment. We have revised the description to clearly state that Wuzhishan pigs are a typical miniature breed. The revisions are highlighted in yellow in L557-566.

 

• Please reduce repetition of villus explanation focus on what is novel for this breed.

AU: We thank the reviewer for the comment. We have reduced repetitive explanations of intestinal villi and focused the discussion on the novel findings for the Wuzhishan pig breed. The revisions are highlighted in yellow in L577-579 and L584-593.

 

• Please fix inconsistency earlier results said ileal α-amylase decreased at maturity, not weaning.

AU: We thank the reviewer for the comment. We have corrected the inconsistency: ileal α‑amylase activity was decreased at maturity, not during weaning, in accordance with the results. The revisions are highlighted in yellow in L599-603.

 

• Author should not recommend enzyme manipulation strategies without testing interventions reframe as hypothesis.

AU: We thank the reviewer for the comment. We have revised the intervention-related statements and reframed them as hypotheses rather than direct recommendations. The revisions are highlighted in yellow in L615-619.

 

• Please avoid claiming beneficial bacteria compensatory mechanism without functional readouts (SCFAs, cytokines).

AU: We thank the reviewer for the comment. We have revised the description to avoid overinterpretation and only state a potential association without claiming a compensatory mechanism. The revisions are highlighted in yellow in L681-685.

 

• Please do not interpret cortisol decrease as causing inflammation cortisol biology is complex and gut luminal cortisol is not equivalent to systemic signaling.

AU: We thank the reviewer for the comment. We have removed the cortisol-related interpretation from the manuscript.

 

• Author should not pool ages within a stage without statistical adjustment this is a major confounder.

AU: We sincerely appreciate the reviewer for this critical and important comment.To avoid confounding effects from age variation within each developmental stage, age was included as a fixed effect in all statistical models to adjust for within‑stage age differences.We have revised the statistical analysis section in the Materials and Methods, and the corresponding content has been highlighted in yellow in Lines 237-238. Our grouping by developmental stages was primarily designed to preliminarily characterize the overall changes in intestinal microbiota and metabolites across different developmental stages of Wuzhishan pigs, while minimizing the interference of age variation within each stage. This design allowed us to first identify key physiological differences among growth stages and determine which critical stages are most relevant to production performance and weight gain, so that we can conduct more detailed and in-depth research targeting these key stages in future studies. We greatly appreciate the reviewer’s professional and valuable suggestion, which has significantly improved the rigor and rationality of our study.

 

• Author should reconcile diet description with lactation/infant stage piglets cannot be fed solid diet in the same way as adults.

AU: We thank the reviewer for the comment. We have revised the diet description to clarify that suckling piglets during the lactation period were fed only breast milk. The revisions are highlighted in yellow in L139-142.

 

• Please specify time from slaughter to freezing, and whether samples were snap-frozen immediately (metabolomics is sensitive).

AU: We thank the reviewer for the comment. We have clarified that intestinal contents were snap-frozen immediately in liquid nitrogen within 3 minutes after slaughter to ensure sample stability for metabolomic analysis. The revisions are highlighted in yellow in L143-145.

 

• Please correct diet tables and confirm nutrient units, current crude fat values are not credible and could invalidate biological interpretation.

AU: We thank the reviewer for the critical comment. We have corrected the unrealistically high crude fat values in all diet tables due to typographical errors:

Table 1: from 33% to 3.3%

Table 2: from 66% to 6.6%

Table 3: from 52% to 5.2%

Table 4: from 23% to 2.3%

The revised contents have been highlighted in yellow in the tables.

 

• Author should clarify whether six villi per section gives enough power and whether villi were randomly chosen.

AU: We thank the reviewer for the constructive comment. We have clarified in the revised manuscript that the villi were randomly selected for measurement. In addition, measuring six villi per section is sufficient to obtain reliable intestinal morphological results, and relevant literature has been cited in the text for support. The revisions are highlighted in yellow in L174-176.

 

• Please correct primer sequences and ensure they match standard 341F/806R variants.

AU: We thank the reviewer for the comment. We have corrected the primer sequences to the standard 341F/806R variants for 16S rRNA gene amplification. The revisions are highlighted in yellow in L193-195.

 

 

• Author should provide exact trimming parameters and rarefaction rationale rarefaction can discard data and alternatives exist.

AU: We thank the reviewer for the valuable comment.We have added the exact trimming parameters in the revised manuscript.For rarefaction, all samples were rarefied to the minimum read count (58,300) to standardize sequencing depth and ensure unbiased comparison of microbial diversity. Although it may discard some reads, this is a widely accepted method to reduce bias from unequal sequencing effort. The revisions are highlighted in yellow in L195-204.

 

Responds to Reviewer 2:

• The manuscript does not fully follow the standard article format of Animals. The Results section is excessively long and unbalanced relative to other sections, and interpretative statements are sometimes included in the Results.

AU: Thank you for your valuable comment. We have carefully revised the manuscript in strict accordance with the standard article format of Animals. We have condensed and rebalanced the length of the Results section, and all interpretative statements originally included in the Results have been moved to the Discussion section. The revisions are highlighted in yellow in L624-632 and 731-761.

 

• The novel contribution is not sufficiently emphasized. Similar microbiome and metabolomic studies in pigs have been reported. The authors should better highlight the breed-specific relevance and the added value of this work.

AU: Thank you for this constructive comment.We have revised the manuscript to better emphasize the novel contribution and breed-specific relevance of this study.Accordingly, we have strengthened the discussion by highlighting the following unique characteristics of Wuzhishan pigs: The stage-specific patterns of protein and fat deposition during weaning, fattening, and maturity; The distinct intestinal microbiota and metabolic profiles related to the breed’s excellent roughage tolerance; The integrated regulation among intestinal morphology, digestive enzymes, microbiota, and metabolomics across different developmental stages. In addition, we have also revised the Conclusions section to emphasize the innovative contributions of this study, highlight the breed-specific significance of Wuzhishan pigs, and further clarify the academic added value of our work.

 

• Experimental and analytical techniques are described without appropriate references. All methods (histology, enzyme assays, sequencing, metabolomics, and statistical analyses) should be fully referenced.

AU: Thank you for your valuable comment. We have added appropriate references to the paragraphs describing all the methods you mentioned, including histology, enzyme assays, sequencing, metabolomics, and statistical analyses. The revisions have been highlighted in yellow at lines 176, 185, 218, and 226.

 

• The exact anatomical locations used for sampling the duodenum, jejunum, ileum, cecum, and colon are not clearly defined.

AU: Thank you for your valuable comment.We have clearly defined the exact anatomical sampling locations for the duodenum, jejunum, ileum, cecum, and colon in the revised Materials and Methods section. The revisions are highlighted in yellow in L145-150.

 

• The Results section includes interpretation and speculation that should be moved to the Discussion. Non-significant trends are largely ignored. 6) The Discussion largely repeats the Results and lacks integrated interpretation across intestinal morphology, enzyme activity, microbiota, and metabolomics. The authors should reorganize this section around biological themes rather than restating results.

AU: Thank you for your valuable and constructive comments.We have revised the Results section by removing the interpretation and speculation, and moved them to the Discussion section as suggested.We have also revised and reorganized the Discussion section according to your comments and those of the other reviewers. The revisions are highlighted in yellow in lines L624–632, L701–719, and L731–761.

 

 

 

Responds to Reviewer 3:

• Abstract: Does not contain any information about materials and methods. It should follow the normal structure of a scientific paper, just short.

AU: Thank you for your valuable comments. We have revised the abstract by integrating your comments and those of other reviewers. The revisions are highlighted in yellow in L34-51.

 

• The sequence of the chapters does not follow the general rules, correct it.

AU: Thank you for your valuable comment. We have adjusted the sequence of the chapters to follow the general rules.

 

• L69-71: This is awkward, not true, revise.

AU: Thank you for your valuable comment. We have revised the inappropriate and inaccurate description in lines 69–71. The revisions are highlighted in yellow in L73-74.

 

• L70: incorrect term “infant period”, it appears also later in the manuscript. In animal science we do not say infant, correct it everywhere.

AU: Thank you for your valuable comment.We have replaced the term “infant period” with “pre-weaning phase” throughout the manuscript.

 

• L83: not correct scientific term: intestinal flora. Flora is reserved to plants; in animals we talk about microbiota. Correct it everywhere.

AU: Thank you for your valuable comment. We have replaced the term “flora” with “microbiota” throughout the manuscript.

 

• L93-95: not only to intestinal cells.

AU: Thank you for your valuable comment. We have revised it accordingly. The revisions are highlighted in yellow in L96-99.

 

• L108-116: The introduction should end with the aims. This part is partly methods, partly conclusions. Should be moved from here.

AU: Thank you for your valuable comment. We have revised the end of the introduction to focus on the research aims, merging and adjusting the content to remove the method- and conclusion-related parts. The revisions are highlighted in yellow in L114-123.

 

• L117: materials and methods should follow the introduction, not the results.

AU: Thank you for your valuable comment. We have revised the manuscript structure accordingly.

 

• L120-: Since the authors are studying a non-traditional breed, age range of the periods should be described to the non-familiar readers.

AU: Thank you for your valuable comment.We have described the age ranges of each developmental stage in the Materials and Methods section before line 120 of the original manuscript. The revisions are highlighted in yellow in L130-32.

 

• Reference to a figure or table should appear after the very first time a data described in the text from the corresponding figure, table.

AU: Thank you for your valuable comment.We have adjusted the position of the figure reference to appear after the first description of the corresponding data in the text. The revisions are highlighted in yellow at Lines 369–372, 435, 444, 528, and 547.

• Figure 1 and all other figures and tables: The term “stages” are incorrect. Use phase, and define age period in days. Sub-figures are not visible, enlarge them. 12) Figure 3: sub-figures are too small, and bad quality, by enlarging the text fall apart, not readable. True to all other figures as well, solve the issue.

AU: Thank you for your valuable comment.We have uniformly revised the term "stages" to "phases" throughout the text, figures, and tables.We have clearly defined each age phase in days when they first appear in the figure captions and table notes.In addition, we have improved the clarity and readability of all figures. The revisions are highlighted in yellow in L163-165 and L291-293.

 

• L158-159: This sentence is logically incorrect, because there are means with double superscript.

AU: Thank you for your valuable comment.We have revised the relevant description throughout the manuscript to ensure logical accuracy. The revisions are highlighted in yellow in Lines 162-163, 295-296, and 311-312.

 

• L282: what roughage tolerance means?

AU: Thank you for your constructive comment.We have clarified and revised the term “roughage tolerance” to the more accurate and standard expression “high-fiber diet tolerance” in the manuscript. The revisions are highlighted in yellow in L405-409.

 

• L432: The breed is a slow growing, small body size genotype, certainly very low nutrient requirement.  So “roughage tolerance” is a reason or a consequence? Discuss it. 16) Discussion: Many parts meaningless general text. Not strong enough. Authors should discuss the protein and fat deposition pattern of the breed.

AU: Thank you for your valuable comments. We have revised the Discussion section based on your suggestions and those from other reviewers. The revisions are highlighted in yellow at Lines 560–561, 593, 615, 624–632, 681–685, 701–719, and 731–761.

 

17)   L582-583: But why? Would not the pigs will grow with the same slow rate?

AU: Thank you for your valuable comment.We have revised the relevant description to clarify that fat supplementation helps improve immunity and intestinal health, thereby minimizing the negative effects of weaning stress on growth and ensuring normal growth performance of Wuzhishan piglets. The revisions are highlighted in yellow in L715-719.

 

18) Conclusions should not be a summary. This part needs to be rewritten. There is not any real conclusion.

AU: Thank you for your comment. We have completely rewritten the conclusion section to highlight the key findings and their implications. The revisions are highlighted in yellow in L763-788.

 

19)  L617-634: methods poorly described, give detailed information.

AU: Thank you for your comment. We have revised the experimental methods as suggested and supplemented more detailed descriptive information. The revisions are highlighted in yellow in L139-150.

 

 

20) Table 1-4: completely incorrect, nutrients are listed as ingredients, no composition data. Statistical analyses: not all are described, correct it.

AU: Thank you for your comments. We have revised Tables 1–4 and improved the description of statistical analyses as suggested. The revisions are highlighted in yellow in L152-159.

 

Response to editor:

• Please add simple summary before abstract. It is vitally important that scientists are able to describe their work simply and concisely to the public, especially in an open-access on-line journal.

AU: We have added a Simple Summary before the abstract as suggested.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a multi-omics investigation of intestinal development in Wuzhishan pigs across four developmental stages, integrating intestinal morphology, digestive enzyme activity, microbiota profiling, and metabolomics. The topic is relevant and the dataset is extensive.

Specific Comments

1. The manuscript does not fully follow the standard article format of Animals. The Results section is excessively long and unbalanced relative to other sections, and interpretative statements are sometimes included in the Results.
2. The novel contribution is not sufficiently emphasized. Similar microbiome and metabolomic studies in pigs have been reported. The authors should better highlight the breed-specific relevance and the added value of this work.
3. Experimental and analytical techniques are described without appropriate references. All methods (histology, enzyme assays, sequencing, metabolomics, and statistical analyses) should be fully referenced.
4. The exact anatomical locations used for sampling the duodenum, jejunum, ileum, cecum, and colon are not clearly defined.
5. The Results section includes interpretation and speculation that should be moved to the Discussion. Non-significant trends are largely ignored.
6. The Discussion largely repeats the Results and lacks integrated interpretation across intestinal morphology, enzyme activity, microbiota, and metabolomics. The authors should reorganize this section around biological themes rather than restating results.

 

Author Response

Dear Editor and Reviewers,

On behalf of the authors of the submitted manuscript “Evaluating Different Stages of Intestinal Development in Wuzhishan Pigs Through Microbiome and Metabolomic Analysis”, we sincerely thank you for your valuable reviews and suggestions. These comments are very helpful. We have carefully studied the comments and made corresponding revisions, which we hope will meet with your approval.The main revisions in the paper (changes highlighted in yellow) and the responses to the reviewer’s comments are as follows:

Responses to the reviewer’s comments:

 

Responds to Reviewer 2:

• The manuscript does not fully follow the standard article format of Animals. The Results section is excessively long and unbalanced relative to other sections, and interpretative statements are sometimes included in the Results.

AU: Thank you for your valuable comment. We have carefully revised the manuscript in strict accordance with the standard article format of Animals. We have condensed and rebalanced the length of the Results section, and all interpretative statements originally included in the Results have been moved to the Discussion section. The revisions are highlighted in yellow in L624-632 and 731-761.

 

• The novel contribution is not sufficiently emphasized. Similar microbiome and metabolomic studies in pigs have been reported. The authors should better highlight the breed-specific relevance and the added value of this work.

AU: Thank you for this constructive comment.We have revised the manuscript to better emphasize the novel contribution and breed-specific relevance of this study.Accordingly, we have strengthened the discussion by highlighting the following unique characteristics of Wuzhishan pigs: The stage-specific patterns of protein and fat deposition during weaning, fattening, and maturity; The distinct intestinal microbiota and metabolic profiles related to the breed’s excellent roughage tolerance; The integrated regulation among intestinal morphology, digestive enzymes, microbiota, and metabolomics across different developmental stages. In addition, we have also revised the Conclusions section to emphasize the innovative contributions of this study, highlight the breed-specific significance of Wuzhishan pigs, and further clarify the academic added value of our work.

 

• Experimental and analytical techniques are described without appropriate references. All methods (histology, enzyme assays, sequencing, metabolomics, and statistical analyses) should be fully referenced.

AU: Thank you for your valuable comment. We have added appropriate references to the paragraphs describing all the methods you mentioned, including histology, enzyme assays, sequencing, metabolomics, and statistical analyses. The revisions have been highlighted in yellow at lines 176, 185, 218, and 226.

 

• The exact anatomical locations used for sampling the duodenum, jejunum, ileum, cecum, and colon are not clearly defined.

AU: Thank you for your valuable comment.We have clearly defined the exact anatomical sampling locations for the duodenum, jejunum, ileum, cecum, and colon in the revised Materials and Methods section. The revisions are highlighted in yellow in L145-150.

 

• The Results section includes interpretation and speculation that should be moved to the Discussion. Non-significant trends are largely ignored. 6) The Discussion largely repeats the Results and lacks integrated interpretation across intestinal morphology, enzyme activity, microbiota, and metabolomics. The authors should reorganize this section around biological themes rather than restating results.

AU: Thank you for your valuable and constructive comments.We have revised the Results section by removing the interpretation and speculation, and moved them to the Discussion section as suggested.We have also revised and reorganized the Discussion section according to your comments and those of the other reviewers. The revisions are highlighted in yellow in lines L624–632, L701–719, and L731–761.

 

 

Response to editor:

• Please add simple summary before abstract. It is vitally important that scientists are able to describe their work simply and concisely to the public, especially in an open-access on-line journal.

AU: We have added a Simple Summary before the abstract as suggested.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Abstract: Does not contain any information about materials and methods. It should follow the normal structure of a scientific paper, just short.

The sequence of the chapters does not follow the general rules, correct it.

L69-71: This is awkward, not true, revise.

L70: incorrect term “infant period”, it appears also later in the manuscript. In animal science we do not say infant, correct it everywhere

L83: not correct scientific term: intestinal flora. Flora is reserved to plants; in animals we talk about microbiota. Correct it everywhere.

L93-95: not only to intestinal cells.

L108-116: The introduction should end with the aims. This part is partly methods, partly conclusions. Should be moved from here.

L117: materials and methods should follow the introduction, not the results.

L120-: Since the authors are studying a non-traditional breed, age range of the periods should be described to the non-familiar readers.

Reference to a figure or table should appear after the very first time a data described in the text from the corresponding figure, table.

L130-132: description of methods mixed with results. The authors should distinguish that clearly, not only here, but in the whole manuscript.

Figure 1 and all other figures and tables: The term “stages” are incorrect. Use phase, and define age period in days. Sub-figures are not visible, enlarge them.

L158-159: This sentence is logically incorrect, because there are means with double superscript.

Figure 3: sub-figures are too small, and bad quality, by enlarging the text fall apart, not readable. True to all other figures as well, solve the issue.

L282: what roughage tolerance means?

L432: The breed is a slow growing, small body size genotype, certainly very low nutrient requirement.  So “roughage tolerance” is a reason or a consequence? Discuss it.

L582-583: But why? Would not the pigs will grow with the same slow rate?

Conclusions should not be a summary. This part needs to be rewritten. There is not any real conclusion.

Discussion: Many parts meaningless general text. Not strong enough. Authors should discuss the protein and fat deposition pattern of the breed.

L617-634: methods poorly described, give detailed information.

Table 1-4: completely incorrect, nutrients are listed as ingredients, no composition data.

Statistical analyses: not all are described, correct it.

Comments on the Quality of English Language

Not the proper scientific terms used, therefore it needs serious improvement.

Author Response

Dear Editor and Reviewers,

On behalf of the authors of the submitted manuscript “Evaluating Different Stages of Intestinal Development in Wuzhishan Pigs Through Microbiome and Metabolomic Analysis”, we sincerely thank you for your valuable reviews and suggestions. These comments are very helpful. We have carefully studied the comments and made corresponding revisions, which we hope will meet with your approval.The main revisions in the paper (changes highlighted in yellow) and the responses to the reviewer’s comments are as follows:

Responses to the reviewer’s comments:

 

Responds to Reviewer 3:

• Abstract: Does not contain any information about materials and methods. It should follow the normal structure of a scientific paper, just short.

AU: Thank you for your valuable comments. We have revised the abstract by integrating your comments and those of other reviewers. The revisions are highlighted in yellow in L34-51.

 

• The sequence of the chapters does not follow the general rules, correct it.

AU: Thank you for your valuable comment. We have adjusted the sequence of the chapters to follow the general rules.

 

• L69-71: This is awkward, not true, revise.

AU: Thank you for your valuable comment. We have revised the inappropriate and inaccurate description in lines 69–71. The revisions are highlighted in yellow in L73-74.

 

• L70: incorrect term “infant period”, it appears also later in the manuscript. In animal science we do not say infant, correct it everywhere.

AU: Thank you for your valuable comment.We have replaced the term “infant period” with “pre-weaning phase” throughout the manuscript.

 

• L83: not correct scientific term: intestinal flora. Flora is reserved to plants; in animals we talk about microbiota. Correct it everywhere.

AU: Thank you for your valuable comment. We have replaced the term “flora” with “microbiota” throughout the manuscript.

 

• L93-95: not only to intestinal cells.

AU: Thank you for your valuable comment. We have revised it accordingly. The revisions are highlighted in yellow in L96-99.

 

• L108-116: The introduction should end with the aims. This part is partly methods, partly conclusions. Should be moved from here.

AU: Thank you for your valuable comment. We have revised the end of the introduction to focus on the research aims, merging and adjusting the content to remove the method- and conclusion-related parts. The revisions are highlighted in yellow in L114-123.

 

• L117: materials and methods should follow the introduction, not the results.

AU: Thank you for your valuable comment. We have revised the manuscript structure accordingly.

 

• L120-: Since the authors are studying a non-traditional breed, age range of the periods should be described to the non-familiar readers.

AU: Thank you for your valuable comment.We have described the age ranges of each developmental stage in the Materials and Methods section before line 120 of the original manuscript. The revisions are highlighted in yellow in L130-32.

 

• Reference to a figure or table should appear after the very first time a data described in the text from the corresponding figure, table.

AU: Thank you for your valuable comment.We have adjusted the position of the figure reference to appear after the first description of the corresponding data in the text. The revisions are highlighted in yellow at Lines 369–372, 435, 444, 528, and 547.

• Figure 1 and all other figures and tables: The term “stages” are incorrect. Use phase, and define age period in days. Sub-figures are not visible, enlarge them. 12) Figure 3: sub-figures are too small, and bad quality, by enlarging the text fall apart, not readable. True to all other figures as well, solve the issue.

AU: Thank you for your valuable comment.We have uniformly revised the term "stages" to "phases" throughout the text, figures, and tables.We have clearly defined each age phase in days when they first appear in the figure captions and table notes.In addition, we have improved the clarity and readability of all figures. The revisions are highlighted in yellow in L163-165 and L291-293.

 

• L158-159: This sentence is logically incorrect, because there are means with double superscript.

AU: Thank you for your valuable comment.We have revised the relevant description throughout the manuscript to ensure logical accuracy. The revisions are highlighted in yellow in Lines 162-163, 295-296, and 311-312.

 

• L282: what roughage tolerance means?

AU: Thank you for your constructive comment.We have clarified and revised the term “roughage tolerance” to the more accurate and standard expression “high-fiber diet tolerance” in the manuscript. The revisions are highlighted in yellow in L405-409.

 

• L432: The breed is a slow growing, small body size genotype, certainly very low nutrient requirement.  So “roughage tolerance” is a reason or a consequence? Discuss it. 16) Discussion: Many parts meaningless general text. Not strong enough. Authors should discuss the protein and fat deposition pattern of the breed.

AU: Thank you for your valuable comments. We have revised the Discussion section based on your suggestions and those from other reviewers. The revisions are highlighted in yellow at Lines 560–561, 593, 615, 624–632, 681–685, 701–719, and 731–761.

 

17)   L582-583: But why? Would not the pigs will grow with the same slow rate?

AU: Thank you for your valuable comment.We have revised the relevant description to clarify that fat supplementation helps improve immunity and intestinal health, thereby minimizing the negative effects of weaning stress on growth and ensuring normal growth performance of Wuzhishan piglets. The revisions are highlighted in yellow in L715-719.

 

18) Conclusions should not be a summary. This part needs to be rewritten. There is not any real conclusion.

AU: Thank you for your comment. We have completely rewritten the conclusion section to highlight the key findings and their implications. The revisions are highlighted in yellow in L763-788.

 

19)  L617-634: methods poorly described, give detailed information.

AU: Thank you for your comment. We have revised the experimental methods as suggested and supplemented more detailed descriptive information. The revisions are highlighted in yellow in L139-150.

 

 

20) Table 1-4: completely incorrect, nutrients are listed as ingredients, no composition data. Statistical analyses: not all are described, correct it.

AU: Thank you for your comments. We have revised Tables 1–4 and improved the description of statistical analyses as suggested. The revisions are highlighted in yellow in L152-159.

 

Response to editor:

• Please add simple summary before abstract. It is vitally important that scientists are able to describe their work simply and concisely to the public, especially in an open-access on-line journal.

AU: We have added a Simple Summary before the abstract as suggested.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been revised in accordance with the reviewers’ comments and the authors have made appropriate efforts to address the previously raised concerns. The revisions have improved the clarity, organization, and overall quality of the manuscript. In its current form the article is considered suitable for publication, as the authors have adequately responded to the comments and incorporated the necessary modifications.

Author Response

Dear Reviewers,   We sincerely appreciate your professional and constructive comments, which have greatly improved the quality of our manuscript. We are honored that the revised manuscript is considered suitable for publication, and we would like to express our heartfelt gratitude again for your rigorous review and invaluable guidance.

Reviewer 2 Report

Comments and Suggestions for Authors

 

• The title could be simplified to improve clarity and conciseness.

• The Simple Summary should be shortened and written in clearer, more accessible language for a broader audience.

• Some figure text is difficult to read due to small font size and/or low resolution; figure quality should be improved.

• Several sections contain repeated or duplicated text and should be revised to avoid redundancy.

• The order of some sections appears inconsistent and should be reorganized.

• Section 5 (Materials and Methods) repeated and should be corrected.

 

Author Response

Dear Editor and Reviewers,

On behalf of the authors of the submitted manuscript “Evaluating Different Stages of Intestinal Development in Wuzhishan Pigs Through Microbiome and Metabolomic Analysis”, we sincerely thank you for your valuable reviews and suggestions. These comments are very helpful. We have carefully studied the comments and made corresponding revisions, which we hope will meet with your approval.The main revisions in the paper (changes highlighted in yellow) and the responses to the reviewer’s comments are as follows:

Responses to the reviewer’s comments:

 

Responds to Reviewer 2:

• The title could be simplified to improve clarity and conciseness.

AU: We thank the reviewer for the constructive suggestion. The title has been revised to improve clarity and conciseness as suggested. The revised title is:Intestinal Development in Wuzhishan Pigs at Different Growth Stages: Insights from Microbiome and Metabolomics. The revisions are highlighted in yellow in L2-3.

 

• The Simple Summary should be shortened and written in clearer, more accessible language for a broader audience.

AU: We thank the reviewer for the constructive suggestion. The Simple Summary has been revised to be shorter and expressed in clearer, more accessible language. The revisions are highlighted in yellow in L19-28.

 

• Some figure text is difficult to read due to small font size and/or low resolution; figure quality should be improved.

AU: We thank the reviewer for the constructive suggestion. We have adjusted all figures to the highest possible resolution. All text in the figures is now clear and legible when viewed at an appropriate magnification.

 

• Several sections contain repeated or duplicated text and should be revised to avoid redundancy.

AU：We thank the reviewer for the constructive suggestions. We have revised the parts with repeated text and eliminated redundancy in the manuscript. The revisions are highlighted in yellow at lines 1919, 2289-2292, 2648, and 2947-2967.

 

• The order of some sections appears inconsistent and should be reorganized. 6) Section 5 (Materials and Methods) repeated and should be corrected.

AU: We thank the reviewer for the constructive suggestions. We have reorganized the order of the relevant sections to ensure logical consistency, and carefully checked and removed any repeated content in Section 5 (Materials and Methods) to ensure the clarity of the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

L141: breast milk is a more human expression, say no prestarter feed was fed. But then, when they have been weaned? How the piglets were adapted to solid feeding? Give details.

L143: Describe procedure of slaughter (immobilization, exsanguination, etc)

Table 1: Also table 2,3,4 : Indicate which values are analysed, which ones are calculated, or from the label of the bag. No information about ingredient composition?

L152: content not composition

L162-163: Over explained, correct it (at every other places as well): Means in a row lacking a common superscript differ (P<0.05). This table is a result, and has no reference here. Should be moved to the proper place.

L238-239: Why? GraphPad can also do this. Since within phases a distant samplings were taken, authors needs to include the sampling time effect in the statistical analyses. Moreover on the figures (for instance figure 1) authors should present the data for each sampling age and indicate that to which phases those ages are falling. In such a way regression equations could be fitted on the data, giving the opportunity of better interpretation of the results.

L249-253: Materials and methods, not results.

L556-557: delete

L559-561:  provide references for these statements

L560: roughage resistance: correct it to high fiber tolerance

L563: Does the data shows, that they can utilize more efficiently of fiber then modern genotypes? discuss it.

L565: present data on daily weight gain.

L570-582: only meaningless text, delete it.

L604-611: contains repetitions, revise.

L663: Lee et al. reference number missing.

L682-684: repetition

L749: Lactobacillus is not a fungus, correct it.

750-785: This is a repetition of the results, not real discussion. Revise. Results should be opposed to modern genotypes more.

L787-790: meaningless, delete.

L792-795: this is true for the modern genotypes as well, what is the new information?

L798-800: meaningless, delete.L

803-812: Sorry to say, but this is a general bla-bla,  nothing concrete. Unfortunately this is true for the whole conclusions. Authors should conclude in 4-5 sentences the concrete new information arise from the research.

L813-880: repetition, delete

L1000-1001: not numbered, not referenced, incomplete.

Comments on the Quality of English Language

Terminology still needs improvement.

Author Response

Dear Editor and Reviewers,

On behalf of the authors of the submitted manuscript “Evaluating Different Stages of Intestinal Development in Wuzhishan Pigs Through Microbiome and Metabolomic Analysis”, we sincerely thank you for your valuable reviews and suggestions. These comments are very helpful. We have carefully studied the comments and made corresponding revisions, which we hope will meet with your approval.The main revisions in the paper (changes highlighted in yellow) and the responses to the reviewer’s comments are as follows:

Responses to the reviewer’s comments:

 

Responds to Reviewer 3:

• breast milk is a more human expression, say no prestarter feed was fed. But then, when they have been weaned? How the piglets were adapted to solid feeding? Give details.

AU: We greatly appreciate the constructive comments and valuable suggestions. We have revised the description regarding the feeding regime of suckling and weaned piglets. Specifically, we have clearly stated that no prestarter feed was provided during the suckling period, and we have supplemented detailed information about weaning age (35 days of age) and the gradual adaptation procedure to solid feed. The revisions are highlighted in yellow in L134-140.

 

• L143: Describe procedure of slaughter (immobilization, exsanguination, etc) Table 1: Also table 2,3,4 : Indicate which values are analysed, which ones are calculated, or from the label of the bag. No information about ingredient composition?

AU: We sincerely appreciate the reviewer’s comments.We have described the slaughter procedure in detail in the revised manuscript and supplemented the information on the company and standards used for analyzing the feed nutritional components. The revisions are highlighted in yellow in L140-151.

 

• L152: content not composition

AU: We thank the reviewer for the correction. "composition" has been revised to "content" as suggested.

 

• L162-163: Over explained, correct it (at every other places as well): Means in a row lacking a common superscript differ (P<0.05). This table is a result, and has no reference here. Should be moved to the proper place.

AU: We appreciate the reviewer’s comments. The over‑explained text has been corrected and simplified as suggested, and the relevant content has been moved to the appropriate position. The revisions are highlighted in yellow at lines 291, 355, 357, and 306.

 

• L238-239: Why? GraphPad can also do this. Since within phases a distant samplings were taken, authors needs to include the sampling time effect in the statistical analyses. Moreover on the figures (for instance figure 1) authors should present the data for each sampling age and indicate that to which phases those ages are falling. In such a way regression equations could be fitted on the data, giving the opportunity of better interpretation of the results.

AU: We greatly appreciate the reviewer’s valuable and constructive comments.The description of the statistical methods has been revised accordingly. The revisions are highlighted in yellow in L745-746. In addition, we sincerely appreciate the reviewer’s rigorous and important comment. To avoid confounding effects caused by age differences within each developmental stage, age had already been included as a fixed effect in all statistical models to adjust for within-stage age variation. The present study was grouped by developmental stage, with the primary aim of preliminarily revealing the overall dynamic patterns of intestinal microbiota and metabolites in Wuzhishan pigs across different developmental stages, while minimizing the confounding influence of age variation within stages. This design allowed us to first identify key physiological differences among growth stages and determine the critical periods most relevant to growth performance and weight gain, supporting more detailed and in-depth investigations targeting these critical stages in future research. Currently, other researchers have conducted in-depth studies based on the present findings, including investigating the specific change patterns of key developmental stages in Wuzhishan pigs at the age level, as well as conducting relevant studies on effective nutritional factor regulation. We are very grateful for the reviewer’s professional and valuable suggestions, which have significantly improved the rigor and rationality of this study.

 

• L249-253: Materials and methods, not results. 7) L556-557: delete

AU: We appreciate the reviewer’s comments. We have moved the content in Lines 249–253 of the original manuscript to the Materials and Methods section and deleted Lines 556–557 as suggested.

 

• L559-561:  provide references for these statements. 8) L560: roughage resistance: correct it to high fiber tolerance. 9) L563: Does the data shows, that they can utilize more efficiently of fiber then modern genotypes? discuss it. 10) L565: present data on daily weight gain.

AU: We appreciate the reviewer’s comments. Relevant references have been added to support the statements in Lines 559–561. The term “roughage resistance” has been corrected to high fiber tolerance. We have further discussed the superior fiber utilization efficiency of Wuzhishan pigs compared with modern genotypes as suggested. The revisions are highlighted in yellow in L558-567.

 

11)  L570-582: only meaningless text, delete it.

AU: We appreciate the reviewer’s comments. The meaningless text in Lines 570–582 has been deleted as suggested.

 

12) L604-611: contains repetitions, revise.

AU: We appreciate the reviewer’s comments. The repetitive content in Lines 604–611 has been revised as suggested. The revisions are highlighted in yellow in L 612-615.

 

13) L663: Lee et al. reference number missing.

AU: We appreciate the reviewer’s comment. The reference number for Lee et al. has been correctly placed as suggested. The revisions are highlighted in yellow in L 638-643.

 

14) L682-684: repetition.

AU: We appreciate the reviewer’s comments. The repetitive content in Lines 682–684 has been removed as suggested. The revisions are highlighted in yellow in L 669.

 

15) L749: Lactobacillus is not a fungus, correct it. 

AU: We appreciate the reviewer’s comment. The mistake that Lactobacillus was referred to as a fungus has been corrected. The revisions are highlighted in yellow in L 715.

 

16) 750-785: This is a repetition of the results, not real discussion. Revise. Results should be opposed to modern genotypes more.

AU: We appreciate the reviewer’s comments. The repetitive content in Lines 750–785 has been revised, and the results have been further compared with modern genotypes as suggested. The revisions are highlighted in yellow in L 718-738.

 

17) L787-790: meaningless, delete. 18) L792-795: this is true for the modern genotypes as well, what is the new information? 19) L798-800: meaningless, delete. 20) L803-812: Sorry to say, but this is a general bla-bla,  nothing concrete. Unfortunately this is true for the whole conclusions. Authors should conclude in 4-5 sentences the concrete new information arise from the research.

AU: Thank you for your valuable comments.We have completely revised the conclusion section into 4–5 concise sentences that only present the concrete new findings of this study, removing all general and empty descriptions. The repetitive content in Lines 750–785 has been revised, and the results have been further compared with modern genotypes as suggested. The revisions are highlighted in yellow in L740-754.

 

21) L813-880: repetition, delete. 22) L1000-1001: not numbered, not referenced, incomplete.

AU: We sincerely apologize. Due to the large discrepancy between the line numbers in the revised manuscript you provided and those you indicated, and the maximum line number of the revised manuscript being 3363, we were unable to locate these two issues. Therefore, we have provided a PDF version of the revised manuscript for your reference.

Author Response File: Author Response.pdf