PONE-D-25-33278 Effect of pre-freezing semen dilution rate and cryo-protection with dimethyl acetamide on post-thaw motility and fertility traits of rooster sperm PLOS ONE

Dear Dr. Gamal M. K. Mehaisen

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PLOS ONE

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Partly

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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: No

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: The manuscript presents a well-designed study addressing an important topic in poultry reproduction. The experimental design is robust, data analysis is appropriate, and conclusions are supported by the results. Minor revisions are required to clarify methodological details and improve data presentation.

1. Check the chart format, the indicators and values do not correspond; Split table presentation is recommended; It is recommended to clarify the number of Hatched chicks, etc;

2. It is recommended to supplement other important experimental results in addition to data, such as pictures;

3. The overall fertilization rate result is low, please analyze and discuss this result;

4. The format of the references does not agree to be unified and there are format errors, please correct them; It is recommended to refer to literature from recent years.

Reviewer #2: Title

• Please consider making the title more precise, for example: “Effect of semen dilution rate and dimethyl acetamide levels on post-thaw motility and fertility parameters of rooster sperm.”

Abstract

• The concluding sentence needs improvement. It should be more practically oriented, emphasizing potential applications in field conditions.

Introduction

• Please highlight the novelty of the study more explicitly.

• Line 49: The phrase “freezing technique per se” is unclear and requires clarification.

Materials and Methods

• Line 113: Specify the initial temperature at the time of dilution.

• Lines 115–118: Provide the source and catalog numbers for all products used.

• Line 119: Clarify the cooling rate, i.e., the time required for lowering the temperature from the initial to the final (5 °C).

• Line 126: Verify and clearly state the temperature and corresponding time duration.

• Line 138: The statement “After dilution with pre-warmed EK extender to a final ratio of 1:50 (v/v)” is unclear. Please rephrase for clarity.

• Line 153: The insemination dose (300 × 10⁶ sperm) requires a reference to justify its selection.

• Line 154: Specify the volume of thawed semen used for each insemination.

Statistical Analysis

• The fertility data is not appropriate for Chi-Square analysis. Logistic regression with a suitable distribution (e.g., Binomial, events/trials) is recommended.

• What was the experimental unit for the fertility data: eggs or hens? Apparently, it looks like eggs, which is not correct. Also, provide the main effects of dose (Low or High concentrations of sperm) or DMA concentrations and interaction, along with polynomial contrast with fertility data.

• Indicate the experimental design used (e.g., split-plot or completely randomized design).

Results

• Lines 180–181: This statement is a repetition of the previous one and should be removed.

• Tables: Place the p-values of main effects, interactions, and contrasts in columns instead of rows, it’ll improve readability.

• Table 1: Please confirm whether “n” for LSC and HSC groups is 54 or 72.

• Table 2: Provide both numerical values and percentages (e.g., for fertile eggs under LSC treatment, report “n” alongside 6.7%).

• Include a correlation table linking motility parameters with fertility outcomes. Try to establish which motility parameters more accurately describe the fertility outcomes.

Discussion

• The discussion requires substantial improvement; it is too lengthy and at times repetitive.

• Address discrepancies observed in the results (e.g., why the HSC resulted in increased early and late chick mortality).

• Elaborate the practical importance of sperm motility parameters (VCL, VSL, LIN, STR, WOB, ALH, BCF) and their relevance for field application.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PONE-D-25-33278.pdf

Effect of semen dilution rate and dimethyl acetamide levels on post-thaw motility and fertility parameters of rooster sperm

PONE-D-25-33278R1

Dear Dr. Gamal M. K. Mehaisen,

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,

Sameh Abdelnour

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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