Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study presents a well-designed and deep insight investigation into Lactobacillus rhamnosus (LR) and Bacillus subtilis (BS) evaluation on an in vitro method. Pretty unique and important to Poultry readers in Animals. The authors demonstrate that Eimeria infection induces microbiome dysbiosis with draft change in microbial metabolome. Although paper is good writing, some minor improvements could be made before publishment.

1. Please improve the flowability of the passage. Some paragraphs are dense with information, but the logical transitions between sentences could be tighter, such as the end of the introduction, parts of the discussion.
2. The topic sentences of some paragraphs are not prominent enough, requiring readers to infer the core point.
3. Some terms and abbreviations are used inconsistently or are not defined upon first use. Such as "Eimeria-disturbed control" and "Eimeria-infected slurry." Consider standardizing to "Eimeria-infected group" or similar. LR and BS, as well as PA, PC, lysoPC.
4. In the results, instead of "Eimeria infection increased alpha diversity" please replace with "Eimeria infection resulted in an increase in microbial alpha diversity …"
5. When describing key findings, consistently cite the relevant figure/table in parentheses at the end of the sentence or at the relevant point.
6. The discussion section could delve deeper into the mechanistic differences between LR and BS. The conclusion that BS efficacy may be more host-dependent is a strong point, but the argument could be further substantiated.
7. Typos: perturbed", "in-fected" with a hyphen, "Eimiera", etc.
   1. Author Contributions (P11, L12-14): The text mentions "Y.N. and X.Y. wrote the first draft," but "Y.N." is not listed in the author list. Please verify and correct the initials.
   2. Figure 5 Legend (P9, L1-6 & Fig. 5): The legend describes red/blue dots for metabolites/microbes and solid/dashed lines for correlations. Ensure these visual elements are distinct and clearly identifiable in the figure as presented.
8. Line 31-32 & 127-128: Why authors choose 3×10⁵ CFU/mL for both probiotics?
9. Line 136-137. How do authors know the system achieved similar colonizer numbers as well as diversity?
10. Figures 2, 4, 5. The words in the pictures are not clear in the print version. Authors may choose to reduce half of the numbers of pictures in each row.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript, titled “Utilizing an in vitro fermentation model to evaluate the effects of probiotics on the cecal microbiota and metabolome disrupted by Eimeria,” describes the effects of two probiotics on restoring the composition of the gut microbiota. Generally, The MS is well written and organized. However, there are several points to consider.

1. It is very unfortunate that the analysis was conducted on a sample collected at a specific point in time (8 days after infection). If you have results from analyzing samples taken at other points in time, please add them.
2. The cecum is influenced by a wide variety of factors within the microbiota. The modified VL medium used in the experiment does not accurately mimic the intestinal environment. Therefore, please describe the shortcomings of this experiment in the discussion section.
3. Cecal microbiota is disturbed mainly during 4 to 6 days after E. tenella infection. Is there any specific reason to use cecal samples collected on day 8 after infection?
4. The intestines are teeming with anaerobic microorganisms, and their influence is also crucial. In line 132, why was aerobic fermentation chosen for this study?
5. can you add sample numbers in Figures (n=??)?
6. To improve comprehension and accuracy, please replace Figure 4 with a table.
7. Add a line name or specific characteristics of the yellow broiler to line 93.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 

The manuscript by Yani Wu et al. is focused on the research of the effects of commensal bacteria (Lactobacillus rhamnosus and Bacillus subtilis) on the cecal microbiome and metabolome from the Eimeria tenella coccidial-infected chickens. The authors' conclusions are consistent with the results obtained, and the manuscript sounds scientific. Nevertheless, I believe the manuscript needs revision.

1. The Introduction section does not contain a stated aim, which would improve the overall presentation of the material.
2. Microbiome section in M&M: There are neither raw sequencing data statistics nor a clear pipeline. Therefore, it is unclear whether the authors filtered low-abundance sequences (singletons) (essential for accurate microbial community profiling), and if so, at what step of bioinformatics pipeline it was done, it is unclear. If they did this after OTU clustering, the diversity indices could have differed dramatically compared to if they had performed this operation before. This clarification about the filtration step of low-abundant sequences should be included in the text of M&M, or the step-by-step processing pipeline might be provided in the appendices.
3. Please clearly state how many samples biological replicates were used per each sample type; this is currently unclear.
4. Figures 2 and 5 need revision, as the font is so small that it is illegible.
5. It is not clear where the raw sequencing data is located, please provide a link to the repository
6. There are typos throughout the text, for example, line 153.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is well responsed accroding to comments.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have provided the responses to all my comments, therefore I do not have any further suggestions.