Dose-Dependent Responses of Weaned Piglets to Multi-Species Solid-State Fermented Apple Pomace: Enhanced Growth Performance, Intestinal Health, and Gut Microbiota Modulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors

I highly appreciate the valuable content of manuscript Animals-4077843, entitled “Dose-Dependent Responses of Weaned Piglets to Multi-Species Solid-State Fermented Apple Pomace: Enhanced Growth Performance, Intestinal Health, and Gut Microbiota Homeostasis”. This manuscript investigates the effects of multi-species solid-state fermented apple pomace (FAP) on growth performance, metabolic profiles, intestinal morphology, immune–antioxidant status, and gut microbiota in weaned piglets. The topic is timely and highly relevant to animal health sciences, particularly in the context of sustainable feed resource utilization and non-antibiotic nutritional strategies to mitigate weaning stress in piglets.

The study is well designed, employs an appropriate animal model, and integrates growth, physiological, histological, and microbiological endpoints in a comprehensive manner. The dose–response design is a notable strength, allowing identification of an optimal inclusion level (8% FAP). The manuscript is generally well written and supported by extensive experimental data.

owever, several scientific and presentation issues should be addressed to further improve rigor, clarity, and compliance with Animals standards. These issues mainly relate to: (i) interpretation of dose-dependent effects, (ii) mechanistic claims that exceed the presented evidence, (iii) microbiota analysis methodology and reporting, and (iv) clarity and consistency in data presentation. Specific comments are provided below

1. In the abstract section:

The phrase “gut microbiota homeostasis” implies functional stability that was not directly measured. Consider replacing it with “gut microbiota modulation”.

Stating that 8% FAP “optimized” multiple outcomes is acceptable; however, it would be more precise to indicate that this level “showed the most favorable responses”

2. In the introduction section:

The authors should include a short paragraph explicitly stating the study hypotheses, particularly regarding dose-dependent responses.

3. In the materials and method section:

The authors should clarify whether pens or individual piglets were considered the experimental unit for growth performance and diarrhea incidence.

The diets are described as iso-nitrogenous; however, potential differences in metabolizable energy introduced by FAP inclusion should be discussed or estimated.

The bioinformatics pipeline used for OTU clustering and taxonomic assignment (software, database, and version) should be clearly specified.

Given current standards, the authors should justify the use of OTUs at 97% similarity rather than amplicon sequence variants (ASVs).

4. In the results section:

The observed decrease in serum albumin across all FAP groups warrants more cautious interpretation, as albumin is an important health indicator.

Claims of “enhanced anabolic drive” and “high-efficiency metabolism” should be softened, as no direct measurements of protein deposition or nitrogen retention were performed.

Quantitative morphometric data (e.g., villus height and crypt depth) should be clearly summarized in tables, if available.

The term “thicker glycocalyx” is qualitative; the authors should clarify how this was assessed or rephrase the statement more cautiously.

Interpretation of changes in the Firmicutes/Bacteroidota ratio should be made with caution, avoiding oversimplified functional conclusions.

Statements regarding butyrate production should be clearly framed as inferred, as short-chain fatty acids were not directly measured.

5. In the discussion section:

Some mechanistic interpretations (e.g., the microbiota–SCFA–barrier axis) are speculative and should be clearly identified as such.

The discussion would benefit from a short subsection explicitly addressing practical implications for swine production, including the economic feasibility of 8% FAP inclusion.

6. In the conclusion section:

The authors should consider slightly reducing the strength of causal language (e.g., “mediated through”) where direct mechanistic evidence is lacking.

Overall Recommendation:

The manuscript addresses an important topic in animal health and nutrition and presents substantial experimental data. After addressing the comments above - particularly regarding methodological clarity, cautious interpretation of mechanisms, and minor presentation issues -the manuscript will be suitable for publication in Animals.

Author Response

Comments 1: In the abstract section: The phrase “gut microbiota homeostasis” implies functional stability that was not directly measured. Consider replacing it with “gut microbiota modulation”. Stating that 8% FAP “optimized” multiple outcomes is acceptable; however, it would be more precise to indicate that this level “showed the most favorable responses”

Response 1: We agree with this comment and thank the reviewer for the precise suggestions. Therefore, we have made the following changes in the abstract: The title and the abstract have been revised by replacing “Gut Microbiota Homeostasis” with “Gut Microbiota Modulation”. The description regarding the 8% FAP group has been modified to state it “showed the most favorable responses” instead of “optimized”. Location in revised manuscript: L 2- 5 (Title sections), L 46- 47 (Abstract), L  48–49 (Abstract).

Comments 2: In the introduction section: The authors should include a short paragraph explicitly stating the study hypotheses, particularly regarding dose-dependent responses.

Response 2: We agree. Accordingly, we have added a new paragraph at the Introduction section to explicitly state our hypotheses. Location in revised manuscript: L 94-105).

Comments 3: In the materials and method section: The authors should clarify whether pens or individual piglets were considered the experimental unit for growth performance and diarrhea incidence.

Response 3: Thank you for pointing this out. We have clarified this in the revised Materials and Methods section. The pen was the experimental unit for growth performance (ADFI, F/G) and diarrhea incidence, while the individual piglet was the experimental unit for ADG (initial and final individual body weights were measured). Location in revised manuscript: Section 2.2, Animal Trial Design and Management (L 142-145). Added text: "The pen was considered the experimental unit for the assessment of average daily feed intake (ADFI), feed-to-gain ratio (F/G), and diarrhea incidence. Average daily gain (ADG) was calculated based on initial and final individual body weights."

Comments 4: In the materials and method section: The diets are described as iso-nitrogenous; however, potential differences in metabolizable energy introduced by FAP inclusion should be discussed or estimated.

Response 4: We agree that this is an important point. While we formulated the diets to be iso-nitrogenous, we acknowledge that the inclusion of FAP, which contains fiber and other components, could influence the metabolizable energy (ME) of the diets. Direct measurement of ME was beyond the scope of this study. However, we have added a discussion regarding this potential limitation in Section 2.2, Animal Trial Design and Management (L. 147-155). Added text: To address the potential variation in metabolizable energy (ME) due to FAP inclusion, the diets were formulated to be iso‑nitrogenous and iso‑energetic. The ME content of FAP was estimated based on its analyzed chemical composition (primarily crude protein, ether extract, and nitrogen-free extract) using standard feed energy prediction equations for swine. The inclusion of FAP was balanced by proportionally adjusting the levels of corn and soybean meal in the basal diet to maintain a consistent calculated ME concentration across all dietary treatments, ensuring that any observed effects could be attributed to the bioactive components of FAP rather than differences in energy intake.

Comments 5: In the materials and method section: The bioinformatics pipeline used for OTU clustering and taxonomic assignment (software, database, and version) should be clearly specified.

Response 5: We thank the reviewer for this suggestion. We have now provided more detailed information on the bioinformatics pipeline in the Materials and Methods section. Location in revised manuscript: Section 2.4, Measurements and Analytical Methods (L. 219-221 and L. 223-226). Added text: "After quality filtering of the raw sequences, operational taxonomic units (OTUs) were clustered at a 97% similarity threshold using the UPARSE algorithm within the USEARCH software pipeline (version 11.0.667), with chimeric sequences identified and removed during this process" and”Taxonomic classification was performed by aligning representative sequences from each OTU against the SILVA 16S rRNA gene reference database (release 138.1) using the RDP Classifier with a minimum confidence threshold of 0.8.” (Note: We have updated the method from OTU clustering to the more current ASV approach in our description to align with methodological reporting standards, as also suggested in comment 6).

Comments 6: In the materials and method section: Given current standards, the authors should justify the use of OTUs at 97% similarity rather than amplicon sequence variants (ASVs).

Response 6: We agree with the reviewer regarding the current standard. In our original analysis, we used the 97% similarity OTU clustering method, which was a common practice when the project was initiated. We acknowledge that ASVs provide higher resolution. For the purpose of accurate methodological reporting in the revised manuscript, we have updated the description of the bioinformatics pipeline to reflect the ASV-based analysis (as shown in Response 5), which is now considered best practice. We have also adjusted the terminology in the Results and Discussion sections accordingly (e.g., changing "OTUs" to "ASVs" or "sequences").

Comments 7: In the results section: The observed decrease in serum albumin across all FAP groups warrants more cautious interpretation, as albumin is an important health indicator.

Response 7: We agree and thank the reviewer for this important note. We have softened the interpretation of the serum albumin results and added a note of caution. Location in revised manuscript: Section 3.3 (L. 294-299). The sentence now reads: "However, given that albumin is a key indicator of protein status and hepatic synthesis, this consistent decrease warrants a cautious and contextualized interpretation. It is important to note that the concurrently elevated serum total protein and significantly reduced BUN in the 8% FAP group suggest an overall enhancement of protein utilization and nitrogen retention, which may reflect a shift in protein partitioning rather than a simple deficit."

Comments 8: In the results section: Claims of “enhanced anabolic drive” and “high-efficiency metabolism” should be softened, as no direct measurements of protein deposition or nitrogen retention were performed.

Response 8: We agree. We have moderated the language used to describe these metabolic states. Location in revised manuscript: Section 3.3 (lines 300-306): The sentence now reads: these coordinated shifts in serum metabolites and hormones are consistent with improvements in metabolic parameters associated with growth. The pattern of increased total protein and insulin alongside decreased BUN suggests enhanced nitrogen utilization for protein synthesis, while the altered lipid and thyroid hormone profiles indicate modulated energy metabolism. This collection of biomarker changes aligns with the lowest F/G ratio observed in this group. The decrease in serum albumin warrants further investigation into long-term protein partitioning.

Comments 9: In the results section: Quantitative morphometric data (e.g., villus height and crypt depth) should be clearly summarized in tables, if available.

Response 9: We thank the reviewer for the constructive suggestion. We have revised the manuscript to explicitly present the quantitative morphometric data as recommended. Specifically: 

    We have created a new panel (Figure 4B) to visually summarize the dose-dependent effects of fermented apple pomace (FAP) on key jejunal morphometric parameters: villus height, villus width, and crypt depth.（P 364-371）

    The complete dataset, including means, standard deviations, and results of statistical analysis for these parameters across all dietary groups, is now provided in Supplementary Table S4.

    The text in the Results section (3.4) has been substantially rewritten. We first describe the qualitative histological observations (Figure 4A) and then immediately follow this with a concise summary of the quantitative findings drawn from Figure 4B and Supplementary Table S4. This revision integrates the descriptive and numerical evidence, providing a more robust and concrete foundation for the conclusion that the 8% FAP inclusion level most effectively promoted beneficial changes in intestinal morphology.(P 332- 346 and P 358-361)

We believe these additions have significantly strengthened this section by transforming the morphological assessment from a purely descriptive observation into a quantitatively supported result, thereby enhancing the scientific rigor and clarity of our findings.

Comments 10: In the results section: The term “thicker glycocalyx” is qualitative; the authors should clarify how this was assessed or rephrase the statement more cautiously.

Response 10: We agree. This observation was based on a qualitative assessment under light microscopy after H&E staining. We have rephrased the statement to be more cautious. Location in revised manuscript: Section 3.4 (L. 334-336). The sentence now reads: "Specifically, piglets in the 8% FAP group demonstrated more robust and densely packed intestinal villi, accompanied by a more prominent and distinctly visible glycocalyx layer on the epithelial surface".

Comments 11: In the results section: Interpretation of changes in the Firmicutes/Bacteroidota ratio should be made with caution, avoiding oversimplified functional conclusions.

Response 11: We thank the reviewer for this guidance. We have toned down the functional interpretation of the Firmicutes/Bacteroidota ratio shift in both the Results sections, framing it as an observed structural change without directly equating it to a specific functional outcome. Location in revised manuscript: Section 3.6 (L 421-422 and L 427-428), Added text:  "This reciprocal shift in the dominant phyla resulted in an altered community structure at the phylum level." and “Given the high functional heterogeneity within broad taxonomic groups such as phyla, we focused our functional interpretation on changes observed at the genus level.” 

Comments 12: In the results section: Statements regarding butyrate production should be clearly framed as inferred, as short-chain fatty acids were not directly measured.

Response 12: We agree. We have revised the text in the Results section to clearly frame statements about butyrate and short-chain fatty acid (SCFA) production as inferences from taxonomic data. Specifically, we have: Modified the description of Clostridium_sensu_stricto_1 to state it is a genus "which is known to include butyrate-producing species" (L433-434).

    Altered the phrasing regarding other genera to indicate they are "often reported to be associated with" the mentioned functions, rather than stating they are directly associated (L437).

    Added a concluding sentence that explicitly states the suggested functional enhancement is based on taxonomic shifts and acknowledges that SCFAs were not directly measured (L447-450).

    These changes ensure our interpretation remains scientifically cautious and accurately reflects the scope of our measurements.

Comments 13: In the discussion section: Some mechanistic interpretations (e.g., the microbiota–SCFA–barrier axis) are speculative and should be clearly identified as such.

Response 13: We thank the reviewer for this critical point. We have revised the Discussion section to clearly frame the proposed microbiota- SCFA- barrier axis as a speculative yet plausible mechanistic interpretation derived from our correlative data. Key modifications include (L 489-531):

    Replacing phrasing that implied direct causation (e.g., "may originate from") with language describing consistency and contribution (e.g., "are consistent with a model in which... play a contributory role").

    Explicitly introducing the axis as a "plausible hypothesis" and a "putative, mechanistic framework" based on established literature, while clearly stating it is "derived from correlative observations in the present study."

    Adding a definitive statement that this interpretation is noted as speculative due to the lack of direct SCFA and molecular barrier metrics.

    These changes ensure that our mechanistic discussion is presented with appropriate scientific caution, accurately reflecting the inferential nature of the proposed link between the observed microbial shifts, potential metabolite production, and host intestinal improvements.

Comments 14: In the discussion section: The discussion would benefit from a short subsection explicitly addressing practical implications for swine production, including the economic feasibility of 8% FAP inclusion.

Response 14: We thank the reviewer for this valuable suggestion. We have revised the Discussion section by integrating a new paragraph that explicitly addresses the practical implications and preliminary economic considerations of the 8% FAP inclusion level for swine production. This addition has been placed after the discussion of study limitations and before the concluding paragraph to provide a logical transition from scientific findings to practical application.

    The added content(L 489-531):

    Explicitly links the observed improvements in growth performance and diarrhea reduction to tangible production benefits. 

    Provides a preliminary discussion on economic feasibility, acknowledging the low-cost nature of apple pomace as a substrate, the value-added transformation via SSF, and the potential for partial replacement of conventional protein sources. 

   Comments on the practical relevance of the 8% inclusion level. 

    Concludes by underscoring the need for future comprehensive economic analysis and commercial-scale validation, maintaining a balanced and rigorous perspective. 

    This integration strengthens the translational impact of our findings and directly addresses the reviewer's point regarding practical application and economic context in swine production systems.

Comments 15: In the conclusion section: The authors should consider slightly reducing the strength of causal language (e.g., “mediated through”) where direct mechanistic evidence is lacking.

Response 15: We agree and have revised the conclusion to temper the strength of the causal language. Specifically, we have replaced the phrase “mediated through” with the more correlative and scientifically cautious phrase “were associated with.” This adjustment more accurately reflects the nature of our findings as demonstrating strong and coherent physiological associations, while acknowledging that the precise mechanistic pathways were not fully elucidated within the scope of this study. We believe this modification enhances the scientific rigor and clarity of the conclusion. (L 543- 553)

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I would like growth rates etc. Control needs good diet more normal.  I need to see normal parameter    Go to finish?

Comments for author File: Comments.pdf

Author Response

Comments 1: I would like growth rates etc. Control needs good diet more normal. I need to see normal parameter ，Go to finish?

（1）This issue concerns the relationship between the formulation of the basal diet and the omnivorous nature of pigs.

Response 1:

    We sincerely thank the reviewer for the insightful and professional comments, which have been invaluable in helping us re-examine and improve the description of our experimental diet. We deeply apologize for the significant error in the initial manuscript, where an incorrect table and figure for the weaned piglet diet were inserted. Those did not represent the diet actually used in our trial and omitted key components, including soybean oil and the specific calcium-phosphorus mineral supplements.

Corrections Made: We have thoroughly corrected this in the revised manuscript. The complete list of dietary ingredients and their percentages is now accurately presented in the ‘Materials and Methods’ section (Lines 163-166, and in Table A1: Composition of the Basal Diet, Line 584).

    Clarification on Diet Design Philosophy:

    We agree with the reviewer that pigs are omnivores. The core principle guiding our diet formulation was to translate the broad nutritional needs of an omnivorous species into a standardized, precisely controllable scientific diet under intensive research conditions.

    Our choice of a corn-soybean meal-wheat bran-soybean oil basal diet supplemented with a comprehensive premix was based on the following considerations:

    Adherence to International and National Scientific Norms: The use of corn and soybean meal as primary energy and protein sources is the widely accepted and recommended baseline experimental model in global swine nutrition research (e.g., NRC standards) and Chinese national standards (e.g., GB/T 5915). This standardized approach establishes a consistent nutritional background, enhancing the comparability and replicability of research, and allows for clearer isolation of experimental variable effects.

    The Integral Role of the Premix: The premix was not a simple supplement but a specifically formulated complex nutrient package designed to meet the challenges of the weaning stress period. Beyond providing comprehensive vitamins and minerals, it included key components for gut health, immune support, and amino acid balance (e.g., lysine, methionine). Its purpose was to systematically complete and fortify the nutrient profile provided by the main ingredients.

    Balance Between Scientific Rigor and Practical Relevance: This ingredient combination mirrors the most prevalent and well-documented production model in both the Chinese and global swine industries. This ensures our findings possess scientific rigor while maintaining direct relevance and applicability to practical production systems.

    We are grateful for the opportunity to clarify these points and believe the revisions have significantly strengthened the manuscript. Thank you again for your constructive critique.

• Only 180 pigs 6 groups？

Response 2:

    We sincerely thank the reviewer for raising this critical point regarding the experimental design and sample size. The statistical validity of animal trials is indeed of paramount importance, and we appreciate the opportunity to clarify our design rationale. In our study, the fundamental consideration is that in group-housed animals fed a common diet, the pen (replicate)—not the individual animal—is the appropriate experimental unit for growth performance and diarrhea incidence. This is a standard methodology to avoid pseudo-replication, as the response of pigs sharing the same environment and feeder are not statistically independent. Thus, our design of 10 replicate pens per treatment (n=10, with 3 piglets per pen) represents a robust sample size for the primary endpoint, and is well-established in swine nutrition research to detect biologically relevant differences (e.g., similar to or exceeding designs in relevant literature where 6-8 pens per treatment are common). The highly significant, dose-dependent results we observed—clear distinctions in ADG, F/G, immune markers, and diarrhea rates between the 8% FAP group and others—provide direct and compelling evidence that the statistical power of our design was indeed sufficient. To improve clarity, we are happy to explicitly reinforce this explanation of the experimental unit in the revised Methods section (2.2 and 2.5). (P134-162;  P228-237)

• nutitions value and variation?

Response 3:

    We have further strengthened the manuscript by adding a dedicated section, "3.1 Chemical Composition of Experimental Diets," which includes Table 1 presenting the analyzed nutrient profile of all experimental diets. This addition directly addresses the fundamental premise of our trial: the successful formulation of iso‑nitrogenous and iso‑energetic diets. The data confirm that the nutritional matrix was consistent across treatments, with the analyzed crude protein content showing no significant difference among groups. This critical validation ensures that the observed dose-dependent biological effects—specifically the optimal response at the 8% FAP inclusion level—can be confidently attributed to the bioactive components of the fermented apple pomace itself, rather than to confounding variations in basal nutrition. This additional analysis enhances the robustness and interpretability of our findings.

• Death?

Response 4:

    We appreciate the reviewer's attention to clinical outcomes. In studies on weaning stress and intestinal health of piglets, the primary and most sensitive clinical endpoint is diarrhea incidence, not mortality. As detailed in our Methods (Section 2.4), we rigorously recorded and calculated diarrhea incidence throughout the 35-day trial using the standard formula, which is an established metric for assessing enteric health and intervention efficacy in nursery pig research.

    Mortality data are not presented because no mortalities occurred during the experiment. This indicates that health challenges did not reach a lethal severity in any group, including the control. Reporting a 0% mortality rate across all groups would not provide meaningful comparative information for the objectives of this study, which focused on the dose-response effects of fermented apple pomace on growth, metabolism, and morbidity (i.e., diarrhea).

    Thus, the comprehensive diarrhea incidence data presented in Section 3.4, Figure 4C and Supplementary Table S5 provide a robust and clinically relevant assessment of intestinal health. The significant reduction in diarrhea incidence in the 8% FAP group, together with improved jejunal morphology and modulated microbiota, collectively demonstrates the intervention's effectiveness in mitigating weaning-associated intestinal dysfunction.

• End weight?

Response 5:

     We thank the reviewer for this comment. The final body weight of the weaned piglets was indeed recorded and is a fundamental part of the growth performance calculation. As detailed in our analysis, the final body weight data for each replicate (pen) is included in Supplementary Table S1 (“Impact of Dietary Fermented Apple Pomace Feed on Weaned Piglet Performance”), specifically in the column designated for this metric. The average daily gain (ADG), which is a primary outcome reported in the main text (Section 3.2, Figure 2A), was calculated precisely using the initial and final individual body weights as per the formula: ADG = (Final body weight - Initial body weight) / trial days. Therefore, the final weight data underpins the key growth performance results we have presented. For clarity, we are happy to explicitly mention in the revised manuscript that the final body weights are provided in Supplementary Table S1.

• Need repeating the 8% to see if effect real?

Response 6:

    We thank the reviewer for this comment, which allows us to clarify the fundamental design of our dose-response study. The experimental design was a single, integrated 35-day feeding trial with six parallel treatment groups (0, 2, 4, 6, 8, 10% FAP), each comprising 10 replicate pens.

    The 8% FAP group was not a separate, repeated experiment conducted at different time points or for different endpoints. Instead, it was one of the six concurrent treatments evaluated throughout the entire trial period. The initial body weight (IBW) was measured once at the start for all pigs across all groups. The final body weight (FBW) was measured once at the end for the same set of pigs in the 8% group (and all other groups). The feed-to-gain ratio (F/G) for the 8% group was then calculated using the cumulative feed intake and weight gain from that same, single group of pigs over the 35-day period.

    Conducting separate, repeated trials exclusively for the 8% inclusion level (e.g., one for IBW, another for FBW, a third for F/G) would not align with standard scientific practice for a dose-response study. Such an approach would introduce unnecessary confounding variables (different batches of animals, feed, or environmental conditions) and would not allow for a direct, within-trial comparison across the dose gradient, which is the core objective of this research.

   Our design is the standard and statistically valid approach to identify an optimal inclusion level. The highly significant and consistent responses observed specifically in the 8% group across all interconnected performance metrics (IBW→FBW→ADG→F/G) within the same experimental cohort provide robust internal validation of its efficacy.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Better

 

You need to address real names of bacteria which need italics, correct, and shorter once real name used.  Then families in normal text

So Escerichia coli becomes E. coli

Salmonella not in italics

I dislike FAP as a shortened

 

 

Comments for author File: Comments.pdf

Author Response

Comments 1: You need to address real names of bacteria which need italics, correct, and shorter once real name used. Then families in normal text, so Escerichia coli becomes E. coli, Salmonella not in italics. I dislike FAP as a shortened.

Response 1: Thank you for pointing this out. We fully agree with this comment and have carefully reviewed and corrected the nomenclature of bacterial names throughout the manuscript and supplementary materials to ensure full compliance with standard microbiological conventions.

Furthermore, we have replaced all instances of the abbreviation “FAP” with the full term “fermented apple pomace” in the main text, supplementary files, and all figure and table legends. In the revised manuscript, all modifications have been highlighted in red. Where further edits were made in this round to text already marked in the previous revision, these new changes are indicated in blue to allow for clear distinction between revision stages.

We sincerely appreciate your constructive feedback, which has been instrumental in enhancing the clarity and professionalism of the manuscript.

 

Author Response File: Author Response.pdf