PONE-D-23-07566Association between blood miR-26a levels following artificial insemination and pregnancy outcome in dairy cattlePLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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

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Reviewer #1: Line 44-45: “Critically, detection of oestrus in dairy farms remains overall inefficient (4)”. This claim is supported by a 20+ year reference. Significant advances in the detection of estrus in bovine females have been made in recent years. For example there are several electronic monitoring systems that improve estrus detection. Use a more recent reference and indicate that even with the evolution of recent years, estrus detection remains a limiting factor for reproductive efficiency.

Lines 47-49: “As a result, opportunities to re-inseminate cows that fail to become pregnant at first or second service are often missed unnecessarily, with important consequences for reproductive efficiency and milk productivity”. Not just the first and second, but after every insemination.

Line 236 “easy to quantify”: What would be the proper definition of "easy to quantify"?. I believe that in this specific case, this is not the case, in view of the complexity described in the material and methods and the authors' description in the discussion text.

Reviewer #2: This is an interesting study evaluating the dynamics of miRNA-26 in blood in pregnant versus non pregnant lactating cows. This is a topic of interest as efforts are continuously made in order to diagnose pregnancy in cattle before 21 days after AI. Although results presented in this manuscript are partly negative, they are completely worth presenting.

I would have some comments to be addressed by the authors before the manscript is accepted for publication:

Abstract: ISG15 is occuring towards the end of the abstract from "nowhere". i consider it is worth introducing this factor before, otherwise it is confusing for the reader.

Introduction: what is known about physiological role of miRNA-26?

Material and Methods: I appreciate the detailed presentation of the RNA analyses by the authors but I find the cell culture part very short and lacking important details. In the way it is presented at the moment, I am not sure if it can be succesfully reproduced. Which type of plates were used? How long? How many samples? How were the cells collected and processed until storage.

Discussion: this part is a little shallow and does not discuss all aspects possibly involved. For example, authors state in the introduction, that most pregnancy losses occur during the first 3 weeks after AI. How can this have affected your results in this study? Also, could it be that plasma is not the ideal sample for detecting changes in miRNA-26 expression?

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Reviewer #1: Yes: Carlos Antônio de Carvalho Fernandes

Reviewer #2: Yes: Dragos Scarlet

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Association between blood miR-26a levels following artificial insemination, and pregnancy outcome in dairy cattle

PONE-D-23-07566R1

Dear Dr. Donadeu,

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,

Muhammad Mazhar Ayaz, Ph.D

Academic Editor

PLOS ONE

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