Unveiling the Kadaknath Gut Microbiome: Early Growth Phase Spatiotemporal Diversity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes original results of gut microbime study in Kadaknath chicken using 16S rRNA metabarcoding. The results are new, correctly analysed and described and manuscript in total might be positively evaluated. However, there are some issues that should be addressed before publishing.

The biggest flaw is related to not structured Introduction and Discussion. Both parts should be improved through structuring in logic sequence of all parts, removal of repeats and exact formulating of the statements. 

For example, the statement from lines 82-84 ("Recent evidence suggests that the microbiome dictates poultry physiology by synthesizing bioactive metabolites, contributing to digestion and absorption, driving energy homeostasis, and maintaining mucosal immune response.") than is repeated with slight changes in lines 89-90, 339-340, 407-408. 

Discussion shoud be shortened by the analysis of the obtained results without repeats of results (e.g., Lines 302-304) and suggestions not relyed on the results obtained. For instance, a statement in lines  344-347 "The temporal and spatial patterns observed in this study provide a valuable framework for designing targeted interventions, such as dietary modifications or probiotic supplementation, to support microbiome development and optimize poultry health." is not determined by the results obtained. Again, such not reliable statements should be corrected or removed, remaining only discussion of results.

Another example - lines 460-462 in Conclusion ("The diverse and specialized microbiota, including high Lactobacillus and Phocaeicola proportions in the ceca, underscores their contribution to disease resistance, nutrient absorption, and immune regulation"). This statement is not conclusion, but suggestion that is not proved by the results obtained. So, the conclusion should be modified seriously. Only reliable conclusions determined by the analysed results shoud be provided here.

Minor revisions.

The title: Dynamics is always studied in the same systems (biological or experimental). However, the authors have studied microbiomes at different stages in different chicken individuals. So, it is not the dynamics, but variability.

In text all Latin names of microbial taxa should be in Italian font according to the International Code of Nomenclature of Prokaryotes. 

 

Author Response

1. The biggest flaw is related to not a structured Introduction and Discussion. Both parts should be improved through structuring in logic sequence of all parts, removal of repeats, and exact formulating of the statements.

For example, the statement from lines 82-84 ("Recent evidence suggests that the microbiome dictates poultry physiology by synthesizing bioactive metabolites, contributing to digestion and absorption, driving energy homeostasis, and maintaining mucosal immune response.") than is repeated with slight changes in lines 89-90, 339-340, 407-408.

Response: To avoid redundancy in the introduction and the discussion, we have extensively revised the text in both sections. This revision includes the sentences mentioned in lines. To enhance readability and maintain a logical flow, we followed the structure describing age results (serially for days 3, 28, and 35), followed by gut regions (serially crop, small intestine, and ceca). 

2. Discussion should be shortened by the analysis of the obtained results without repeats of results (e.g., Lines 302-304) and suggestions not relyed on the results obtained. For instance, a statement in lines 344-347 "The temporal and spatial patterns observed in this study provide a valuable framework for designing targeted interventions, such as dietary modifications or probiotic supplementation, to support microbiome development and optimize poultry health." is not determined by the results obtained. Again, such unreliable statements should be corrected or removed, remaining only discussion of results.

Another example - lines 460-462 in Conclusion ("The diverse and specialized microbiota, including high Lactobacillus and Phocaeicola proportions in the ceca, underscores their contribution to disease resistance, nutrient absorption, and immune regulation"). This statement is not a conclusion but a suggestion that is not proved by the results obtained. So, the conclusion should be modified seriously. Only reliable conclusions determined by the analyzed results should be provided here.

Response:  The Discussion section has been extensively revised and shortened. In the updated version, we have strictly focused on the results observed in this study. All speculative and suggestive statements have been removed to ensure the discussion is data-driven.

Minor revisions.

1. The title: Dynamics is always studied in the same systems (biological or experimental). However, the authors have studied microbiomes at different stages in different chicken individuals. So, it is not the dynamics, but variability.

Response: To reflect the study design accurately, we have revised the title (and manuscript text) to replace "dynamics" with "diversity" to align with the methodology and findings.

2. In text all Latin names of microbial taxa should be in Italian font according to the International Code of Nomenclature of Prokaryotes.

Response: corrected at necessary places.

Reviewer 2 Report

Comments and Suggestions for Authors

The methods part was not well clarified in the abstract, the authors have to insert the treatments and the size of the collected sample.

The part of the statistical analysis is not inserted in the study, it is very important to insert the way of the analysis for the obtained results in a separate part, for example, the Alpha Diversity Analysis (Shannon index, Simpson index,….) and if you used Beta Diversity Analysis.

Please insert the full names of all abbreviations at their first mentions (e.g., GDP in line 42,….).

Line 53, which part is rich in antioxidants (e.g. meat, egg,…).

Line 109: Again, the description of the groups (number, weight,…), their housing and feeding systems, and whether they received any feed supplements such as probiotics, prebiotics, or vaccines is not inserted. However, the authors mentioned a previously described protocol, but every study has to be stand-alone, so please insert such information through the manuscript.

For the results part, the comparisons are confused, you have two main objectives comparisons among ages and comparisons among three GIT sites, so it may be better to divide the results according to this issue.

Line 386, which pathogens?

Line 387, low at which age or site?

Line 403, but here is no probiotic supplementations were done in this study!

Line 465, which strategies! Which result that you obtained can confirm this suggestion?

 

Author Response

1. The methods part was not well clarified in the abstract, the authors have to insert the treatments and the size of the collected sample.

Response: The abstract has been updated, adding information about the methodology and samples detail as “This study utilized the V3-V4 region of the 16S rRNA gene sequence to characterize the spatiotemporal variability and shift in the abundance and composition of the gastrointestinal microbiota in Kadaknath, a high-value indigenous Indian chicken breed. To assess microbiome composition and bacterial abundance shifts, three chickens per growth stage (3, 28, and 35 days) were sampled, with microbiota analyzed from three gut regions (crop, small intestine, and ceca) per bird.”

 

2. The part of the statistical analysis is not inserted in the study, it is very important to insert the way of the analysis for the obtained results in a separate part, for example, the Alpha Diversity Analysis (Shannon index, Simpson index,….) and if you used Beta Diversity Analysis.

Response: We added a separate method section, “​​2.5 Statistical analysis”. The results are now described in separate paragraphs in the section “3.2. The Gut Microbiome of Kadaknath Exhibited Diversification and Maturation with Age.”

 

3. Please insert the full names of all abbreviations at their first mentions (e.g., GDP in line 42,….).

Response: Added. The sentence now read as “...,  and nutrition and contribute annually to the global ‘gross domestic product’ (GDP) [1].”

 

4. Line 53, which part is rich in antioxidants (e.g. meat, egg,…).

Response: The sentence is now updated to  “The meat of this breed, especially the muscle tissues, is rich in antioxidants”

 

5. Line 109: Again, the description of the groups (number, weight,…), their housing and feeding systems, and whether they received any feed supplements such as probiotics, prebiotics, or vaccines is not inserted. However, the authors mentioned a previously described protocol, but every study has to be stand-alone, so please insert such information through the manuscript.

Response: A complete section, “​​2.1. Animal maintenance and sample collection” describing growth conditions and other possible details, is now added to the Methods. No external bioactive substances were fed to the chicks.

 

6. For the results part, the comparisons are confused, you have two main objectives comparisons among ages and comparisons among three GIT sites, so it may be better to divide the results according to this issue.

Response: The interwoven description of the age and gut region comparisons are now extensively resolved. In each section of the results, we first described data for the age (serially for days 3, 28, and 35), followed by gut regions (serially crop, small intestine, and ceca). 

 

7. Line 386, which pathogens? Line 387, low at which age or site?

Response: The sentence is updated to “Besides, although we observed pathogens such as Klebsiella, Campylobacter, and Clostridium, in different organs and time-point the overall proportion of pathogenic genera was low, and no clinical symptoms were observed in the chickens studied.”

 

9. Line 403, but here is no probiotic supplementations were done in this study! Line 465, which strategies! Which result that you obtained can confirm this suggestion?

Response: We acknowledge that the statements regarding ‘potential improvement through probiotic supplementation’ (line 403) and ‘probiotic-based intervention strategies’ (line 465) were speculative and not directly supported by our study results. To ensure accuracy and avoid unsupported claims, we have removed these statements from the revised manuscript.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes original results of gut microbime study in Kadaknath chicken using 16S rRNA metabarcoding. The results are new, correctly analysed and described and manuscript in total might be positively evaluated. All issues has been addressed during revising. However, there are some small revisions are necessary before publishing.

Lines 20-22. The phrase seems awkward and would be better reformulated as follows. "In this study the spatiotemporal diversity of the gastrointestinal microbiota, shifts in taxonomic composition and relative abundances of the main bacterial taxa was characterised in Kadaknath, a high-value indigenous Indian chicken breed, using sequencing of the V3-V4 region 16S rRNA gene."

Line 175. The phrase is not correct and should be changed as follows. "The raw reads of 16S rRNA gene..."

 

Author Response

The manuscript describes original results of gut microbime study in Kadaknath chicken using 16S rRNA metabarcoding. The results are new, correctly analysed and described and manuscript in total might be positively evaluated. All issues has been addressed during revising. However, there are some small revisions are necessary before publishing.

Response: We thank you for reviewing our manuscript. 

Lines 20-22. The phrase seems awkward and would be better reformulated as follows. "In this study the spatiotemporal diversity of the gastrointestinal microbiota, shifts in taxonomic composition and relative abundances of the main bacterial taxa was characterised in Kadaknath, a high-value indigenous Indian chicken breed, using sequencing of the V3-V4 region 16S rRNA gene."

Response: We appreciate your suggestion and update the text suggested.

Line 175. The phrase is not correct and should be changed as follows. "The raw reads of 16S rRNA gene..."

Response: The text is updated to “The raw reads of 16S rRNA”.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors made all of our comments, and the manuscript is now improved and can be accepted for publication

Author Response

The authors made all of our comments, and the manuscript is now improved and can be accepted for publication.

Response: We thank you for reviewing our manuscript.