PONE-D-23-00717Experimental facility had a greater effect on growth performance, gut microbiome, and metabolome in weaned pigs than feeding diets containing subtherapeutic levels of antibiotics: a case studyPLOS ONE

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2. Partial research funding for conducting animal experiments was provided by Land O'Lakes/Purina and managed by BdR. Additional funding provided by Land O'Lakes/Purina to the University of Minnesota was managed by GCS for metabolomics and microbiome analysis and Research Assistantship support for MPT. BdR, TK, and HT from Land O' Lakes/Purina were involved in study design, data collection and analysis, decision to publish, and preparation of the manuscript". This information should be added to the COI/FD statement at a future check.

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"Partial research funding for conducting animal experiments was provided by Land O' Lakes/Purina and managed by BdR. Additional funding provided by Land O' Lakes/Purina to the University of Minnesota was managed by GCS for metabolomics and microbiome analysis and Research Assistantship support for MPT. 

BdR, TK, and HT from Land O' Lakes/Purina were involved in study design, data collection and analysis, decision to publish, and preparation of the manuscript"

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Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: In this paper, the authors address the effect of experimental facility and antibiotic feeding on the growth performance, microbiome, and metabolome of weanling piglets. While antibiotics are a known cause of enhanced animal growth performance, the mechanism remains unknown, so this paper is an important question in the field. The manuscript is clear and well-written with a thorough discussion. See below for detailed commentary:

-Data availability: States the data is fully available but not access information is provided. Apologies if I overlooked it, but please provide public accession numbers

-L25: "on" should be "in the" or something similar to fix grammar

-L79: I appreciate the listing of operating protocols, but unless there is something unusual about the SOPs at this location, this is not needed. Please keep the IACUC information, but the SRUs could be eliminated or more information should be provided in supplemental.

-L112-115: I greatly appreciate the honesty about infectious outbreaks in each experiment. It would be useful to know if S. suis was found in any of the microbiome samples from animals not eliminated from the studies. This could be altering their growth and microbiome (sub-clinically).

-DNA extraction: did you utilize any bead beating with this kit? If so, which type of bead? rpm? time?

-L151: There is much detail provided for the metabolomics, which is great, but was this done previously anywhere? If so, it should be cited. Or is this a modification of a previous protocol?

-L194: Which version of R was used?

-L195: which version of vegan?

-L216/221: one instance says "qiime2" and one says "Qiime 2". Please make consistent throughout the manuscript.

-L216: Which version of Qiime2?

-Tables 4 and on are very different formatting from tables 1-3. Please make consistent

-Figures 1, 2, and 4: For these ordinations, please provide % on each axis

-Figure 3 legend: please italicize p value

-Figures 1-4: It would be useful to include n values in the figure legends for reader clarity.

Reviewer #2: The topic is of relevance and the experimental design and methodology are sound. However, there are several issues that the authors should address. See below.

The statement about the databanke where the raw sequencing data were deposited is missing.

Abstract

Biomarker bacteria for AB treatment were identified. Those should be provided in the abstract.

Metabolites that were differently abundant across experiments/facilities/ages or for AB treatment should be provided in the results section of the abstract.

Also, dimension of changes for differently abundant parameters and growth performance should be provided in the abstract.

Introduction

The authors should elaborate more on how the investigation of the gut microbiome based on 16S rRNA sequencing and serum metabolome can help understanding the mechanisms how AGP work. The authors should provide the rationale for the selection of the gut sites and serum metabolomics.

Both the hypothesis and objective of the experiment needs improvement. Provide the gut sites for the microbiome and for the metabolome and also serum for the metabolome.

Results:

Regarding the presentation of data, there were three experiments with several growing phases. In addition, there was the AB treatment. Please organize the paragraphs in the results section accordingly. Otherwise, colleagues that do not carefully read the M&M section are lost.

Dimension of differences should be provided as % or fold-changes for all investigated paraemeters.

L247-250 Split sentence in 2 or 3 sentences and explain the observed effects separately for the different growing stages.

For metabolomics/microbiome, add the age of the pigs or the time postweaning when samples were collected.

L258 clear separation

Discussion

L311 Provide the length of the experimental phase postweaning.

L317 be more specific about the age-response; explain the mechanisms behind the observation that piglets grew differently when treated with AB.

When discussing the amino acid levels in serum, begin with explaining changes that may have occurred at gut level. Consider that AB could have upregulated the transporter expression at the gut mucosa. Check if amino acids that were differently abundant in serum utilize similar transporters. Also, consider an interaction of AB with the gut microbiome that due to an alteration in its composition may have interfered in amino acid uptake. Are there potential associations between metabolome effects and microbiome effects of the AB treatment.

L357-367 Re-write and shorten this paragraph. Changes in alpha diversity do not always occur. There were compositional changes which are more important to discuss.

Do not discuss data based on phylum level. Discuss data at family or genus level

Table and figures should be self-explanatory. Check that all abbreviations were defined at their first mentioning in the text. Provide the information whether data were across experiments and/or treatment groups in the table headings and figure legends.

Tables have different formats. Format tables according to the author guidelines.

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Reviewer #1: Yes: Katie Lynn Summers

Reviewer #2: No

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Experimental facility had a greater effect on growth performance, gut microbiome, and metabolome in weaned pigs than feeding diets containing subtherapeutic levels of antibiotics: a case study

PONE-D-23-00717R1

Dear Dr. Shurson,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Michael H. Kogut, Ph.D.

Academic Editor

PLOS ONE

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