PONE-D-19-28260

Misadjustment of diurnal expression of core temperature and locomotor activity in lactating rabbits associated with maternal over-nutrition before and during pregnancy

PLOS ONE

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Please read the reviews below and respond accordingly. In particular, however,  there should be less emphasis on the circadian aspect of the paper, as it appears that many aspects of the clock and clock function are not the focus of the reported investigation, even though it does fit with previous studies focusing on the circadian regulation. Additionally, there should be more detail on the statistical analysis.. The manuscript also has some grammatical errors, which should be remedied.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: No

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

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript investigates a novel question using a unique experimental model. The experiment appears to have been well controlled and made key observations of behavior and body temperature rhythms. The next obvious step is investigation of the molecular clock. The authors clearly state this and do not over-interpret the data.

Resolution of some of the figures is very poor and impossible to read some of the smaller figures.

I am not sure Table 1 needs to be reported or could be a supplement, but I will leave that to the editor and author to decide.

741: Access

Reviewer #2: General Comments:

This paper measures daily patterns of locomotor activity and body temperature, along with body temperature. The paper appears to be a companion paper to another submitted paper that measured body weight and fertility, based on the citation in the methods. While the paper focuses on an interesting area of fetal programming, I have several concerns about the experimental methods and presentation of results. Specifically, the paper focuses heavily on the circadian aspect of this experiment, but there is really no examination of any circadian rhythms or circadian clock gene expression. Additionally, there is no description of how acrophase, the only response related to daily rhythms, was determined. Overall, the statistical analysis was poorly performed, and P values of most responses were not presented. Several clarifications must be made, and an overhaul of the statistics and presentation of results must be made before I can consider accepting this paper for publication. Additionally, of the results presented only a small number of them are actually related to the objective of studying the effects of maternal nutrition on diurnal rhythms, and most are related to metabolism (UCP1 and CIDEA expression and day vs. night concentration of blood metabolites). The title and introduction, and objectives of the paper should more accurately reflect the responses measured.

Line-by-line comments:

Abstract:

43- Please more clearly describe the ‘anticipatory component” of body temperature or use different terminology. Same for “energetic components of the rhythm”. The meaning of these phrases is non-obvious

Introduction:

117 – Replace with “Women of reproductive age”

120- Be more specific about what conditions these women exhibit

123- Within the introduction there must be justification for why rabbits are used as a model species for maternal overnutrition, and specifically why they can be a good model for humans, since research in human subjects is directly described in the introduction

134 – There is a lot of information in the introduction describing the effects of maternal overnutrition on the circadian clock. However, there is no measurement of the circadian clock gene expression or circadian rhythms of energy metabolism within this paper. There should be a better connection between how the research presented in this paper relates to the circadian clock, or this information should be removed and the introduction should focus more on behavioral and body temperature outputs

143- A hypothesis must be provided

144 – Objective does not include anything about UCP1 or CIDEA expression levels or blood metabolites, so why were these measured? Please add objectives related to these outcomes. Also, circadian rhythms in behavioral and physiologic markers weren’t measured. In order to characterize a true circadian rhythm, responses must be measured in consistent lighting (i.e. 24 dim light). Please refer to the rhythms measured (really only locomoter activity) as a diurnal or daily rhythm.

156 – Must state the experimental design in the methods

164 – Please provide the total kcal/g of each diet

166 – Replace elaborated with a more appropriate word. Could just say HFCD contained SD supplemented with…

170 – Why where # of does unevenly distributed between treatments. Please provide justification for sample size with power calculation.

173 – Please justify that human chorionic gonadotropin would not affect fetal metabolic programming

178 – Just to clarify- HFCD was really fed -8 to -2 week prior to mating, SD is fed for two weeks prior to mating, and HFCD was fed after mating? To me, this is OK, but it must be made much clearer during the description of the treatments.

177 – Please mention explicitly that you there is a companion paper with the same experimental design that measured fertility, body weight, etc. Also, I hope that the body weight presented in Figure 1 is also not reported in the other paper.

194 – Please define that litter is the experimental unit

200 – How were the subsample of pups chosen for measurement?

217 – Replace “breastfeeding” with either “nursing” or “suckling” here and elsewhere.

218 – Replace “produce” with “stimulate”

251 – Be specific about which behavior and physiologic recordings were made

255 – Why was the lactation period only P12 to P31. Shouldn’t it be P1 to P31?

265 – mRNA levels were not determined by extraction. RNA was first extracted and then mRNA expression of UCP1 and CIDEA were determined by RT-qPCR. Correct wording

290 – Duration and intensity of the anticipatory component of are never defined. Please discuss how this was determined because it is completely unclear

288 – You report acrophase, but there is no description of how this is determined. Determination of acrophase must have been performed using cosinor rhythmometry but there is no description of this is the methods. Cosinor rhythmometry should be performed on data where a diurnal response is measured – so for body temp. and for locomotor activity.

292 – Time course data should be analyzed as repeated measures analysis with day as a repeated factor. A two

302 – GraphPad Prism

Results

336 – Replace “ close similarities” with “no difference”

337 – Replace “plasmatic level” with “plasma concentrations” here and elsewhere

Figure 1A – Clarify that this is birthweight within the figure

Figure 5 – Clarify that this is body temperature

Figure 7 – Repsonse variable is not clear from this figure. Figures should be labeled more clearly. Also, it is not explained in the methods how acrophase was determined

Figure 9 – There is no label of which bars represent UCP1 and which represent CIDEA.

Throughout – P-values should be presented both in text and in figures

Discussion

Discussion was not reviewed because of problems with methods and presentation of results.

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Reviewer #1: No

Reviewer #2: No

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Misadjustment of diurnal expression of core temperature and locomotor activity in lactating rabbits associated with maternal over-nutrition before and during pregnancy

PONE-D-19-28260R1

Dear Dr. Ivette Caldelas,

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

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Academic Editor

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Additional Editor Comments (optional):

I have gone through the comments from the reviewers and the responses from the authors. I think authors have revised their ms according the comments and suggestion from the editor and reviewers.

I recommend this ms can be accepted for publication in Plos One.

Reviewers' comments: