Review Reports - Metabolomic Analysis of Wooden Breast Myopathy Shows a Disturbed Lipid Metabolism

Round 1

Reviewer 1 Report

Dear Authors,

In my opinion the aim of the paper is interesting, but due to methodological and formal aspects I cannot accept this paper for publication.

The paper is chaotic and needs a major rewrite.

Materials and Methods section should be clear and concise. Lab methodologies used should be sufficiently detailed for an independent researcher to repeat the experiment. In the manuscript submitted for evaluation there are many inaccuracies in the paper. For example in the L: 107-109, the Authors report that ‘Out of a total of 51 samples, 14 samples (27%) received a WB score of severe. Moderate WS was observed in 24 samples (47%), and severe WS in 8 birds (16%). Eleven birds exhibited both WB and WS (of which two severe WS).’

Comments: 14 samples and 24 samples and 8 samples is 46 samples. 5 samples are missing. Question: exactly how many samples were analysed in the 'normal', WB and WS group?

Moreover, there is no description of the 'separate trial’ in the Materials and methods section (see L: 264-265 ‘To corroborate this hypothesis, we measured vit. K2 in birds collected in a separate trial (47 birds, of which 15 had WB’...).

There is also lack of consistency in the defining of groups (normal, healthy). Moreover, the first time you use an abbreviation you should explain it and then consistently use only one of the forms throughout your work.

In the 'statistical methods', the Authors state that ‘Data were also analyzed by ANOVA using WS, WB, and their interaction....’ L: 104-105 - Analysis of variance (ANOVA) is a collection of statistical models and their associated estimation procedures (such as the "variation" among and between groups) used to analyze the differences among means. For two groups the t-test should be used. If the authors use analysis of variance (ANOVA), it should be specified whether it is a one-way ANOVA or multi-factor ANOVA. For multi-factor ANOVA, specify which main effects are being analyzed as well as the interactions between them. In addition, please complete how the normality of data distribution and homogeneity of variance were tested.

Another point is the presentation of the results. Results should be clear and concise. Provide a precise description of the experimental results, their interpretation as well as the experimental conclusions that can be drawn. L: 121-126 - these are not results. This is part of the materials and methods section. The results section lacks a description of the results of figures 3-5 (which have been moved to the Discussion section). Moreover, L: 139-142 - What about the level of Ca, Fe, Zn and Co?

Without precise methodology and correctly presented results, it is impossible to draw accurate conclusions about the phenomenon under study.

In turn, the Discussion section is not the place to present results, but to discuss them (see L: 235-240; 250-253; 264-267).

Author Response

Please see the attached files for our feedback to the reviewer

Author Response File: Author Response.pdf

Reviewer 2 Report

Myopathies have risen strongly in recent years, and It’s a nice study by authors that explored Metabolomic analysis of wooden breast myopathy in a chicken model. However, I will suggest some minor points for improvement before the final publication.

-Institutional affliction of the second author is missing.

-For all author please provide institutional email.

-Formatting required.

-Double check the references in the texts and citation section and vice versa.

-Line 46. The incidence of these conditions has been estimated to lead to an economic loss of more 46
than $200 million/per year in the United States (Kuttappan et al., 2016). Please specify “these conditions.”

-Line 61. Wooden breast fillets also show use abbreviation WB is ok.

-Line 76, statistics of choosing 308 male chickens. And please explain the reason why only males have been selected in this study.

-Please explain why Out of a total of 308 samples, only 51 muscle samples were selected.

-Please state clearly how many muscle samples were used for Metabolomic analysis.

-Figures 3 to onwards should be mentioned in the results, not in the discussion section.

Author Response

Please see the attached files for our feedback to the reviewer

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors conducted a metabolomic study on boiler muscle with WS and WB. The results provided some insights into the potential etiology of WB. I have some comments on the structure of the manuscript:

1. Method section:

· Please reorganize the whole section into different sub-sections, such as experimental animals, sample preparation, mass spec condition, ICP-MS condition, mass spectra data processing and multivariate data analysis, statistics, etc.

· Was feed intake measured in the study? Please also provide the feed nutrition information. To draw a conclusion that lipid malabsorption is a potential causative reason for WB, detailed information on feed intake is needed

· Line 121: “…bias was removed by the pre-processing steps described before”. Please add citation

2. Some descriptions in the discussion should be moved to the result section. Pathway analysis is an important part of this study. Figures and texts describing its results in greater detail are needed in the result section. Details are as follow:

· Fig. 1. The authors intended to describe the multivariate data analysis in this figure. Because of this, it needs to show how marker metabolites are picked from this model in a better way.

o Please use label multiple graphs with A, B, C, etc., and define them in the legend. The major graph is a combination of score plot and loadings plot, which is less commonly used. Rather than using this graph, the authors can consider putting the score plot as panel A, the loadings plot and the magnified portion as panel B, as the major graph in the current version contains too much information and can be hard to read

o How did the authors draw the blue dots in the major graph? What do they mean?

o Please consider also putting the volcano plot described in line 125 into Figure 1. In the score plot, the separation of groups is not quite obvious, so it is hard to identify the markers for each group through the loadings plot. The volcano plot can show the markers in a better way.

o Figure 3 can be merged into Figure 1, if the authors feel the volcano plot is not a good idea or simply want to focus on WB-affected metabolites. But this figure needs to be in the result section, as well as lines 235-240. The bar plot for p-values is not needed. The authors can add * to the column of ratio to indicate statistical significance. The whole point is to make the marker identification process from the multivariate data analysis more straightforward to the readers.

o If possible, please provide a better-resolution image

· Lines 133-138. Are taurine and hypotaurine metabolism removed from the individual pathway visualization? Please put the visualization for the major affected pathways that will be discussed (e.g.: arginine and proline metabolism) in the main text as the new Figure 2 or consider moving Table S2 into the main text. Please also extend the description of the pathway analysis result, such as moving lines 155-160 into the result section.

· Figure 2. Please label statistical significance (*) on top of the columns

· Line 151: “…PCA analysis in the current study…”, but Fig.1 shows PLS-DA result.

· Figure 4 and Figure 5, together with their description, need to be moved to results.

Author Response

Please see the attached files for our feedback to the reviewer

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The present paper can be accepted for printing in Metabolites with minor corrections.

L: 118 - Please complete which main effects were analysed.

Please clarify whether the data presented in L: 124-127 are mean and SD, SE or SEM? Please note that the notation of ‘mean ± SD’ although very popular is inappropriate. Arithmetic mean is a measure of location, and standard deviation is a measure of variation. They measure completely different characteristics of a sample and there is no point in linking them with the ± sign. The proper notation is mean ± SEM (or just SE).

Author Response

L: 118 - Please complete which main effects were analysed.

WS x WB is added in the text

The data are presented as mean ± SEM; this is clarified in the text

Reviewer 3 Report

Thank you for the revision. Version 2 looks overall good to me. I like alternative figure 1 a lot better. Please use this one in the main text. I only have two comments:

1. In either section 2.3 data analysis or Figure 1 legend, please give a very brief introduction about the function of the Y loadings plot.

2. Please move Figure 1 a little to the left. The PDF file is not showing the whole graph.

Author Response

Thank you for the revision. Version 2 looks overall good to me. I like alternative figure 1 a lot better. Please use this one in the main text. I only have two comments:

Thanks for your feedback; we have deleted the original figure 1.

In either section 2.3 data analysis or Figure 1 legend, please give a very brief introduction about the function of the Y loadings plot.

A brief explanation has been added: This plot uses the same layout as plots A and C and shows how the response variables (WB and WS) associate with the individual animals (plot A) and the various metabolites that survived the very important parameter selection (plot C)

Please move Figure 1 a little to the left. The PDF file is not showing the whole graph.

Fixed