Intestinal Microbiota Structure of Xichuan Black-Bone Chickens and Preliminary Evaluation of a Probiotic-Based Fecal Microbiota Substitute

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents a comprehensive study on the structure of the intestinal microbiota of Xichuan black‑bone chickens (XBC) and a comparative evaluation between traditional fecal microbiota transplantation (FMT) and a designed probiotic substitute (FMST). The topic is pertinent and of clear interest to avian microbiology, intestinal health, and animal production. However, the work exhibits conceptual, methodological, and editorial deficiencies that must be addressed to meet the standards of a high‑impact journal such as Microorganisms.

To begin with, the manuscript does not include evidence of ethical approval or a statement confirming review by an institutional committee—an essential requirement for studies involving live animals. The absence of this information compromises the seriousness of the experimental protocol and limits the possibility of considering the work as fully valid research in terms of animal welfare and international regulatory compliance.

Furthermore, although the study provides valuable data on the segmented distribution of the intestinal microbiota, the identification of native probiotic strains, and the physiological and immunological comparison between FMT and FMST, the interpretation of the results tends to be excessively broad. Several sections extrapolate mechanisms without direct experimental evidence, leading to speculative conclusions that should be moderated.

From a methodological standpoint, the experimental design is generally adequate, yet it presents important limitations—particularly regarding the development and management of the birds. The metagenomic analysis was conducted on only five adult animals, a number insufficient to capture the natural variability of avian microbiota, especially in a native breed with distinct physiological characteristics. The statistical power is limited and does not allow the establishment of robust patterns. In the FMT/FMST experiment, although each group includes 30 birds, the microbiota analyses are performed on a reduced number of samples, which again weakens the strength of the conclusions. Additionally, the study combines females and males without analyzing the potential effect of sex, a factor that can significantly influence microbial composition and immunological responses.

Regarding presentation, the structure of the manuscript aligns with the format of Microorganisms, but the writing requires professional linguistic revision, preferably by a native speaker of scientific English. Issues of fluency, inconsistent terminology, and grammatical constructions that hinder readability are evident. The editorial quality must be improved to ensure clarity and precision.

The conclusions of the manuscript are consistent with the results presented, but they should be expressed with greater caution. The study demonstrates that the cecum is the segment with the highest microbial richness and that native probiotic strains with functional potential can be isolated. Nevertheless, the manuscript asserts effects and applications that cannot be substantiated with the available data. The claim that FMST may replace FMT requires long‑term trials, robust evaluations of productive performance, and deep functional validation—including metabolic analyses, stability of the probiotic consortium, and effects on production parameters.

In summary, the manuscript addresses a relevant and novel topic but presents significant limitations: reduced sample size, lack of deep functional analysis, speculative discussion in certain sections, and the need for greater statistical and methodological rigor. Overall, the study provides valuable information, but it requires strengthening of the methodology, analysis, and writing to reach the scientific and editorial standards expected in Microorganisms.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

I believe the English in the manuscript can certainly be improved; however, I am not the appropriate person to make detailed linguistic observations, as English is not my native language. Being a Latin American researcher, I recognize the limits of my own linguistic expertise, and therefore I cannot provide the level of editorial evaluation that a native scientific editor would offer.

Author Response

We sincerely appreciate your thorough, constructive and insightful comments on our manuscript Intestinal Microbiota Structure of Xichuan Black-bone Chickens and the Effects of Fecal Microbiota Substitute Transplantation. Every suggestion has been carefully reviewed, and we have revised the manuscript point-by-point as detailed below.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

#General assessment

This manuscript investigates the intestinal microbiota structure of Xichuan black-bone chickens (XBCs). It provides a preliminary evaluation of a fecal microbiota substitute prepared from three cultured strains, relevant to poultry microbiome research and safer microbiota alternatives.

However, the manuscript has major scientific and methodological weaknesses. The central conclusion that FMST can effectively replace traditional FMT is not adequately supported. The study lacks sufficient experimental detail, donor characterization, longitudinal sampling, growth-performance outcomes, pathogen-screening details, functional validation of the selected strains, and robust statistical reporting. There are also inconsistencies in data presentation, overinterpretation of microbiome associations, and insufficient evidence for the proposed mechanism involving Mediterraneibacter and Blautia.

The manuscript currently presents initial findings rather than a comprehensive validation of FMST as a superior alternative, encouraging the audience to view results as early insights.

#Major Comments

1. The title contains typographical and conceptual problems

Title - The title suggests a broad evaluation of “effects” of FMST, but the study assesses only limited short-term intestinal indicators. It does not assess growth performance, feed efficiency, pathogen resistance, mortality, long-term colonization, or production traits.

Suggested revised title: Intestinal Microbiota Structure of Xichuan Black-bone Chickens and Preliminary Evaluation of a Probiotic-based Fecal Microbiota Substitute

2. Lines 17–20; lines 445–453

The manuscript concludes that the prepared FMST can effectively replace traditional FMT and is a safer or superior strategy, but current data do not fully support this claim. The study lacks evidence of functional complexity, colonization effects, or long-term ecological impacts of FMST compared to FMT.

The authors should revise this to: “FMST showed potentially beneficial effects on inflammatory markers and tight-junction protein expression compared with FMT under the conditions tested.”

The claim of replacement should be avoided unless broader functional equivalence is demonstrated.

3. Lines 76–80; lines 121–128

The FMT group received raw fecal microbiota solution, the FMST group received a three-strain preparation, and the CK group received saline. However, the manuscript does not describe:

• donor selection for FMT;
• whether donor feces came from adult XBCs or another source;
• donor health screening;
• pathogen screening;
• antimicrobial-resistance screening;
• exact preparation method of fecal suspension;
• storage and viability of FMT material;
• whether total CFU, bacterial biomass, DNA concentration, or functional activity standardized FMT and FMST;
• whether administration was performed only for 3 days or repeated later;
• when outcome samples were collected after treatment.

Without detailed information on donor selection, fecal suspension preparation, and standardization, the comparison between FMT and FMST may seem unclear, which could undermine the audience's trust in reproducibility and interpretation.

4. Lines 112–120; lines 212–219

The FMST preparation consists of Lactobacillus crispatus, Weissella paramesenteroides, and Bacillus amyloliquefaciens. The manuscript states that strains with “good growth performance” and “stable genetic characteristics” were selected, but no functional screening is reported.

The authors should provide strain-level evidence for:

• acid tolerance;
• bile-salt tolerance;
• adhesion ability;
• antimicrobial activity;
• enzyme production;
• antibiotic-resistance profile;
• hemolysis;
• virulence genes;
• genome sequencing or at least safety screening;
• compatibility among the three strains;
• survival during storage;
• survival after oral administration.

Calling these strains probiotic is premature without evidence of functional benefits and safety, which is essential to build confidence in their potential use.

5. Lines 211–219; lines 333–379

Only eight pure strains were isolated from cecal contents, and three were selected as probiotics. This is a narrow cultivation effort and cannot be assumed to represent the most functionally important cecal bacteria of XBCs. Also, the study states that two pathogenic strains were identified and not recommended for fecal microbiota solution preparation, but this raises questions about whether raw FMT material also contained such undesirable organisms.

The authors should clarify:

• how many colonies were screened initially;
• why only eight pure strains were obtained;
• how the three strains were selected beyond growth performance;
• whether selected strains were isolated from all birds or only one donor;
• whether the selected strains were present in the FMT donor material;
• whether these strains actually colonized chicks after administration.

6. Lines 61–75; lines 172–190

Only five healthy adult male XBCs were used to characterize microbiota across intestinal segments. This is a small sample size for metagenomic profiling, especially if the authors intend to infer breed-specific microbiota structure.

Moreover, only adult males were used for intestinal-structure analysis, whereas the transplantation experiment used one-day-old chicks of both sexes. Adult intestinal microbiota cannot be directly used to interpret early-life chick microbiota development without caution.

The authors should soften breed-level conclusions and clearly distinguish adult segmental microbiota profiling from chick intervention outcomes.

7. Lines 81–98

The manuscript states that metagenomic sequencing was performed, but important details are missing:

• number of reads per sample;
• clean reads after filtering;
• host-read removal rate;
• assembly statistics;
• N50;
• number of predicted genes;
• taxonomic classification method;
• database used for species-level annotation;
• abundance estimation method;
• whether rarefaction depth was standardized;
• how viral reads such as Siphoviridae were handled;
• whether the data are deposited in a public database.

Without these details, the metagenomic analysis is not reproducible.

8. Lines 172–190

The manuscript reports thousands of “species” per intestinal segment, with 7593.2 species in the cecum and 6647.0 in the rectum. These numbers are unusually high for metagenomic species-level assignments in chicken intestinal contents and may reflect gene catalog entries, OTUs, contigs, or database-level taxa rather than validated species.

The authors must define exactly what “species number” means. If these are metagenomic taxonomic bins or database annotations, the term “species” may be misleading.

The table should clarify whether these are: observed species-level taxonomic assignments; OTUs; metagenomic species clusters; nonredundant genes assigned to species; MAGs; Kraken/MetaPhlAn-style taxa; and another metric.

9. Lines 166–170; lines 180–191; lines 229–237

The statistical section states that data are expressed as mean ± SD, but Table 1 and Table 2 footnotes state mean ± SEM. This inconsistency must be corrected.

The authors should report:

• whether data are SD or SEM;
• exact sample size for each analysis;
• exact p-values;
• test statistics;
• whether assumptions of ANOVA were tested;
• whether microbiome data were transformed before ANOVA;
• whether multiple testing correction was applied.

Duncan’s multiple comparison test is relatively liberal. For microbiome and multi-marker data, the authors should consider more conservative approaches or justify this choice.

10. Lines 129–136; lines 203–210; lines 220–246

The manuscript reports alpha diversity, beta diversity, Venn diagrams, and dominant genera, but does not provide robust statistical outputs.

Please add:

• PERMANOVA results for beta diversity;
• PCoA axis variance explained;
• alpha-diversity p-values;
• differential abundance analysis with multiple-testing correction;
• effect sizes;
• confidence intervals;
• rarefaction curves or sequencing-depth normalization.

Claims of “significant differences” among groups are not sufficiently supported without these statistics.

11. Lines 220–246

The FMT group has the highest observed species and Chao1 indices, while FMST has the lowest Shannon index among the three groups. The manuscript later concludes that FMST is superior, mainly due to inflammatory and tight-junction markers. However, the microbiota data alone do not show that FMST reconstructs or replaces FMT-like community diversity.

The authors should separate: microbiota diversity outcomes; intestinal barrier outcomes; inflammatory marker outcomes; inferred health effects.

FMST may reduce inflammation compared with FMT, but that does not mean it replaces FMT microbiologically.

12. Lines 76–80; lines 229–237

The manuscript defines the control group as CK, but Table 2 uses NC. Please standardize group names throughout the manuscript.

13. Lines 193–199; lines 324–337

The manuscript labels Corynebacterium xerosis, Alistipes sp. CAG:831 and Helicobacter brantae as pathogenic bacteria, while several lactobacilli are labeled probiotics. This binary classification is oversimplified and may be misleading.

Some taxa may be commensal, opportunistic, strain-dependent, host-dependent, or annotation artifacts. Species-level identification from metagenomic data should also be interpreted cautiously.

The authors should avoid broad labels such as “pathogenic bacteria” and “probiotics” unless supported by strain-level evidence or virulence testing.

14. Lines 46–49; lines 300–337

The Introduction states that exploring microbiota structure is important for understanding high disease resistance. The Discussion suggests that XBC disease resistance may be related to high probiotic abundance. However, the study does not compare XBCs with other chicken breeds, does not challenge birds with pathogens, and does not measure disease resistance.

This claim should be softened to: “The identified microbiota may provide candidate microbial resources for future studies of XBC health traits.”

15. Lines 259–273; lines 380–397

The manuscript reports that FMT increased IL-6, TNF-α, and IL-1β compared with CK and FMST, suggesting subclinical inflammation. This is an important result, but the manuscript does not provide enough information about the FMT donor material, pathogen screening, endotoxin burden, or timing of tissue collection.

The authors should clarify whether the inflammatory response is due to: pathogen transfer; microbial overload; immune stimulation by diverse microbiota; donor-recipient incompatibility; feed or fecal residues; endotoxin or other microbial products; and early-life immune maturation.

The current explanation that unoptimized FMT caused inflammation is plausible but not proven.

16. Lines 145–165; lines 259–279

Section 2.9 describes ELISA for inflammatory factors. Section 2.10 is titled tight junction protein expression, but qPCR includes IL-6, TNF-α, IL-1β, claudin-1, ZO-1, and caspase-3. This mixes inflammatory, barrier, and apoptosis markers.

The Methods should be reorganized clearly: ELISA for cytokine protein levels; qPCR for inflammatory, barrier, and apoptosis-related genes; and Western blot for claudin-1, ZO-1, caspase-3, and β-actin.

17. Lines 274–279; lines 397–406

The manuscript states that claudin-1 and ZO-1 were lower in FMT and higher in FMST, while caspase-3 was higher in FMT. However, the visible text does not provide numerical values, fold changes, Western blot quantification, or normalization details.

Please provide:

• representative blots;
• densitometry values;
• normalization to β-actin;
• biological replicate number;
• exact p-values;
• qPCR results if corresponding mRNA was measured;
• whether results were repeated independently.

18. Lines 247–258

The manuscript states that the FMT group had the highest V/C ratio, but the difference was not significant. It also states that ileal villi were mostly normal in both FMT and FMST groups. Therefore, the histological evidence does not strongly support major treatment effects.

The authors should not overstate intestinal morphology improvement. They should report exact villus height, crypt depth, V/C ratio, p-values, and representative images with scale bars.

19. Lines 241–243; lines 402–443; lines 445–450

The manuscript concludes that divergent regulation of Mediterraneibacter and Blautia is the primary mechanism underlying differences between FMT and FMST. This is not demonstrated.

The study reports association, not mechanism. No SCFA measurements, isolate experiments, causal perturbation, mediation analysis, or functional metagenomics are provided.

The authors should revise: “represents the primary mechanism” to: “may be associated with the observed differences.” To support the mechanism, the authors would need SCFA quantification, correlation analysis, functional pathway analysis, or targeted manipulation of these genera.

20. Lines 420–436; lines 445–450

The Discussion and Conclusion state that enrichment of Mediterraneibacter and Blautia enhances SCFA production and suppresses inflammatory signaling. However, SCFAs were not measured. Please either measure acetate, propionate, and butyrate in cecal contents or remove causal claims about SCFA-mediated mechanisms.

21. Lines 220–237; lines 380–382

The FMT group has numerically higher observed species and Chao1 values, but the manuscript does not clearly report whether these differences are statistically significant. Also, the CK group has similar Shannon and Simpson indices. The authors should state whether FMT significantly improved alpha diversity or only showed a numerical increase.

22. Lines 121–128; lines 129–165

The manuscript says chicks were administered treatment for three consecutive days. Still, it does not clearly state at what age or how many days after treatment the cecal contents, ileal tissue, ELISA, qPCR, and Western blot samples were collected. This is essential for interpreting whether the effects are acute, transient, or stable.

23. The manuscript lacks production-relevant outcomes. Because the manuscript frames FMST as useful for poultry production, it should ideally include: body weight gain; feed intake; feed conversion ratio; diarrhea or morbidity; survival; immune organ indices; pathogen resistance; longer-term follow-up. Without these, practical claims for poultry production should be softened.

#Minor Comments

1. Lines 17–20

“Effectively replace traditional FMT” is too strong. Replace with “showed potential as a safer standardized alternative under the tested conditions.”

2. Lines 25–45

The breed description is useful but should be more concise.

3. Lines 62–64; lines 76–80

The experiment includes both FMT and FMST. Consider calling it the “transplantation experiment.”

4. Lines 62–64; lines 100–104

Clarify whether the adult males used for microbiota profiling were also the source of the cecal contents used to isolate probiotic strains.

5. Lines 82–86; lines 140–165

Use consistent spacing around units, e.g., 0.2 g, 0.8 mL, 50 mg, 3 μm.

6. Lines 84–85; lines 159–165

Verify the English company name. It may be Beyotime Biotechnology.

7. Lines 92–98

The manuscript states PE150 or PE250. Please specify which sequencing mode was actually used.

8. Lines 96–98

The manuscript says MEGAHIT or IDBA-UD. Please state which assembler was used, or whether both were used for different samples.

9. Lines 102–104

LB is not usually the primary medium for strict lactic acid bacteria. Clarify the rationale for using both MRS and LB.

10. Lines 212–219

Provide strain IDs, GenBank accession numbers, and phylogenetic tree details.

11. Lines 180–191

The table footnote says SEM, while statistical methods say SD. Correct this.

12. Lines 229–237

Use CK instead of NC, or define NC.

13. Lines 333–337

“All three strains have been confirmed to have excellent probiotic properties” is too strong for strains isolated in the current study unless the authors performed probiotic assays. Revise to “These species have been reported to possess probiotic potential.”

14. Lines 280–443

The Discussion contains an extensive literature review about the three selected strains. It should be shortened and focused more directly on the study’s results and limitations.

15. Lines 445–453

The conclusion is too mechanistic and too definitive. It should emphasize preliminary evidence and the need for further validation.

Author Response

We sincerely appreciate your thorough, rigorous, and constructive comments on our manuscript  “Intestinal Microbiota Structure of Xichuan Black‑bone Chickens and the Effects of Fecal Microbiota Substitute Transplantation”. Every concern has been carefully reviewed. Corresponding revisions have been implemented in the revised manuscript, including title modification, text rewriting, supplementary methodological details, moderating over‑stated conclusions, correcting inconsistent notations, and adding explicit limitation statements. Below is our point‑by‑point response.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I would ask the following questions:

1.Why were only these three bacterial strains chosen?

2.What criteria were used in their selection?

3. Is there evidence that they successfully colonize the gut after transplantation?

4. Why does FMT result in higher microbial diversity but FMST is considered an equivalent substitute?

5. Are there any performance data (growth, FCR, mortality)?

6. Were claudin-1 and ZO-1 protein levels measured or only mRNA?

7. Was correction for multiple statistical comparisons performed in the metagenomic analysis?

Comments for author File: Comments.pdf

Author Response

We greatly appreciate your careful evaluation and thoughtful questions regarding our manuscript. Below are our point‑by‑point responses. All corresponding revisions have been implemented in the revised manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

After examining the revised version of the manuscript, it is evident that the previously identified observations were addressed with rigor and precision. The document now demonstrates a strengthened structure, a well‑substantiated argumentation, and a level of writing consistent with scientific and editorial standards. Accordingly, I consider the manuscript to meet the necessary conditions for publication.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

I appreciate the opportunity to review the manuscript. However, I must respectfully note that my evaluation does not extend to the stylistic or linguistic aspects of the text. My comments are limited to the scientific content and methodological considerations, and therefore I am unable to provide an assessment of the writing quality or language usage.

Author Response

We sincerely appreciate your careful re‑evaluation and positive assessment of our revised manuscript. Your detailed and constructive critical comments in the first round were extremely valuable for improving the methodological rigour, logical reasoning, and overall presentation of this work. We are very grateful that you consider the revised manuscript satisfies the publication requirements of "Microorganisms".