Impact of In Ovo Leptin Injection and Dietary Protein Levels on Ovarian Growth Markers and Early Folliculogenesis in Post-Hatch Chicks (Gallus gallus domesticus)
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsWhile the paper investigates the impact of leptin and neonatal nutritional intake on early-life ovarian functions in genetically bred broiler breeder hens, there are areas that could be enhanced for clarity and completeness:
LP an NP should be clarified in the abstract
why the authors have applied 2 way statisitcal analysis on this paper. Please explain in the relevant portion. The statistical analysis methods and results are not clearly presented
why the growth data has not been provided?
conclusion is excessively lengthy, it should be minimized and should be based on the results obtained. also why the feed composition table has not been give?
The paper introduces numerous gene markers and their expression changes, but there's a lack of thorough interpretation of these findings.
The paper inconsistently uses abbreviations such as LP and NP for dietary protein levels without clear explanations.
The paper does not discuss potential limitations of the study, such as the choice of specific genes, the relevance of the chosen age points for analysis, or the generalizability of the findings to other breeds.
Author Response
While the paper investigates the impact of leptin and neonatal nutritional intake on early-life ovarian functions in genetically bred broiler breeder hens, there are areas that could be enhanced for clarity and completeness:
Response: We appreciate the reviewer for reviewing our manuscript thoroughly. We have revised the manuscript considering the reviewer’s comments and suggestions. All correspondences for the comments and others are described below.
LP and NP should be clarified in the abstract.
Response; Thank you for the comment. The acronyms were fully defined, and we have changed NP to SP which stands for standard protein as stated in the abstract (P1 L26).
why the authors have applied 2 way statistical analysis on this paper. Please explain in the relevant portion. The statistical analysis methods and results are not clearly presented.
Response: We used two-way ANOVA to analyze the impact of both leptin and/or dietary protein on the expression of genes in the collected tissues and explain it in the statistical analysis section (P5 L181).
why the growth data has not been provided?
Response: Thank you for the notification. The growth data was prepared as supplementary figure 1 and stated in the P11 L352.
conclusion is excessively lengthy, it should be minimized and should be based on the results obtained. also why the feed composition table has not been give?
Response: Thank you for the valuable suggestions. We rewrote and minimized the conclusion as suggested (P12 L407). The feed composition table is provided as Supplementary Table 1 (P3 L132).
The paper introduces numerous gene markers and their expression changes, but there's a lack of thorough interpretation of these findings.
Response: Thank you for the comment. We explained the functions of studied genes in the introduction (P2 L86-101) and enriched the discussion where information was available in the literature. Data was interpreted for AMH, caspase 3, and GDF9 in P10 L318, FSHR, CYP19A1, GDF9, WNTs, and IGF‑1 in P11 L340. Leptin effects were explained in P11 L373.
The paper inconsistently uses abbreviations such as LP and NP for dietary protein levels without clear explanations.
Response: Thank you for the notification. We clearly explained these acronyms in the materials method section and stated them as; low protein (17% low crude protein; low protein (LP)) or standard protein (22% standard crude protein (SP)). LP and SP were clearly explained in the materials method section (P3 L129). The changes were made in the manuscript accordingly.
The paper does not discuss potential limitations of the study, such as the choice of specific genes, the relevance of the chosen age points for analysis, or the generalizability of the findings to other breeds.
Response: Thank you for the valuable comments. As suggested by the third reviewer, we explained in the introduction why the specific genes and age points were selected for this experiment (P2 L86-100), (P3 L 106)
We discussed the limitations of the study and the generalizability of the findings in the discussion section (P12 L397)
Reviewer 2 Report
Comments and Suggestions for AuthorsThis is a good paper planned to assess whether in ovo leptin and post-hatch protein intake levels affect the functional development of the ovary in broiler chicks to seek a possibility to prevent showing PCOS-like phenotype in adulthood.
The topic is of interest for poultry reproduction and health studies. A significant amount of data have been reported. The findings have been properly discussed by using relevant literature.
However, some revisions and improvements may add further value to the manuscript.
Here some suggestions:
- Try to reduce both the Simple Summary and Abstract;
- Check for typos into the full-text;
- Check all the acronyms used if they were spelled at first use;
- In the M&M section, please add references related to the methods used to collect the different data and for procedures;
- The Conclusions section should be improved;
- Check if all references have been cited into the text or reported into the list.
Moderate editing of English language required.
Author Response
This is a good paper planned to assess whether in ovo leptin and post-hatch protein intake levels affect the functional development of the ovary in broiler chicks to seek a possibility to prevent showing PCOS-like phenotype in adulthood.
The topic is of interest for poultry reproduction and health studies. A significant amount of data have been reported. The findings have been properly discussed by using relevant literature.
However, some revisions and improvements may add further value to the manuscript.
Response: We appreciate the reviewer for reviewing thoroughly our manuscript, and thanks for considering our work valuable. The manuscript has been revised and improved in light of the reviewer’s comments and suggestions as follows.
Here some suggestions:
- Try to reduce both the Simple Summary and Abstract;
Response: Thank you for the comment. We concise the summary and abstract.
- Check for typos into the full-text;
Response: We appreciate the suggestion, the manuscript was checked thoroughly for grammar and typos by the authors and the English editing service.
- Check all the acronyms used if they were spelled at first use;
Response: Thank you for the notification, we fully defined the acronyms and explained the function of genes selected for this study in the introduction, as it was also suggested by other reviewers.
- In the M&M section, please add references related to the methods used to collect the different data and for procedures;
Response: Thank you for the comment. In the revision, we cited several new papers regarding materials and methods and highlighted them.
- The Conclusions section should be improved;
Response: Thank you for the important comment. The conclusion was revised.
- Check if all references have been cited into the text or reported into the list.
Response: We used Zotero software based on journal suggestions and manually cross-checked all the references throughout the manuscript.
Comments on the Quality of English Language
Moderate editing of English language required.
Response: Thank you for the suggestion, the manuscript was revised by the authors and edited by an English editing service for appropriate grammar and typos.
Reviewer 3 Report
Comments and Suggestions for AuthorsLine 122. Mention egg sealing methodology in the manuscript.
Author Response
Line 122. Mention egg sealing methodology in the manuscript.
Response: Thank you for the comment. We sealed the pinhole with cellophane tape (P3 L121).
Reviewer 4 Report
Comments and Suggestions for AuthorsThe paper is well-written and pleasant to read. The authors should carefully check to define all the gene symbols and define (write the full name) them before usage.
I suggest explaining the roles of investigated genes in the introduction section to allow the reader to easily comprehend the results thus clarifying why they selected these genes for this experiment. Lastly, they refer to women PCOS in the paper. Are those genes relevant also in human research? I think in this context maybe citing women's PCOS is misleading, also because, it's not clear if their results are useful in any way to women's PCOS. At the end of the study they only state that maybe protein intake is important in young humans. I suggest sticking with chickens.
LINE 14: Define PCOS
LINE 26-44: The abstract should be simplified. I think mentioning all the genes regulated in the abstract confuses the reader.
LINE 84: CYP19 (aromatase cytochrome p450) and other gene abbreviations should be written as full names and defined at first usage.
LINE 87: remove the uppercase T
LINE 102 and line 104: Remove the repeated "we hypnotized" .
LP and NP should be explained in the figures' captions. Moreover, +/- SE of the mean is misleading. It's better to write: "data are reported as Mean +/- SE" or something similar.
In conclusion, the results are interesting. In the future could be fascinating to perform this study on lying hens and compare the results.
The paper is well written and authors should check for minor misspellings or errors.
Author Response
Comments and Suggestions for Authors
The paper is well-written and pleasant to read.
Response: We would like to express our sincere appreciation to the reviewer for their insightful and thorough review of our manuscript. The constructive feedback and thoughtful comments have significantly enriched the quality of our work.
The authors should carefully check to define all the gene symbols and define (write the full name) them before usage. I suggest explaining the roles of investigated genes in the introduction section to allow the reader to easily comprehend the results thus clarifying why they selected these genes for this experiment.
Response: Thank you for the valuable suggestions. We have now fully defined the acronyms at first use and explained the function of genes and the reason for their selection in the introduction (P2 L86-L101).
Lastly, they refer to women PCOS in the paper. Are those genes relevant also in human research? I think in this context maybe citing women's PCOS is misleading, also because, it's not clear if their results are useful in any way to women's PCOS. At the end of the study they only state that maybe protein intake is important in young humans. I suggest sticking with chickens.
Response: Thank you for the important comments and suggestions. We revised the conclusion section in the manuscript, focusing specifically on chickens.
Even though, our results might not be directly associated with women's PCOS. But the genes such as AMH, LH, and FSH are known to be associated with irregular follicular hierarchy in adult chickens (P2 L90). Other genes examined in the study are regulated directly or indirectly by gonadotropins, AMH, or synergistically controlling ovarian functions with leptin and IGF-1 (P2 L92-L101).
LINE 14: Define PCOS
Response: Thank you for the notification, PCOS was defined as [polycystic ovary syndrome (PCOS)] in the Simple Summary (P1 L12).
LINE 26-44: The abstract should be simplified. I think mentioning all the genes regulated in the abstract confuses the reader.
Response: Thank you for the suggestion, the abstract was simplified. The words “Ovarian growth markers essential for folliculogenesis” were used instead of mentioning the names of the genes (P1 L29).
LINE 84: CYP19 (aromatase cytochrome p450) and other gene abbreviations should be written as full names and defined at first usage.
Response: Thank you for the notification, we fully defined the acronyms at first use throughout the manuscript and highlighted them.
LINE 87: remove the uppercase T
Response: Thank you for the notification. The correction was made and checked all through the manuscript.
LINE 102 and line 104: Remove the repeated "we hypnotized".
Response: Thank you for the comment. We corrected and edited these lines for better clarity (P3 L103-L105).
LP and NP should be explained in the figures' captions. Moreover, +/- SE of the mean is misleading. It's better to write: "data are reported as Mean +/- SE" or something similar.
Response: Thank you for the suggestions. We changed The NP to SP which stands for standard protein and changes were made throughout the manuscript. Both acronyms were defined in all figures' captions and stated as; LP (low crude protein, 17%), SP (standard crude protein, 22%) in figure legends.
The suggested correction was made and stated in the figures' captions as follows. The data represent the mean ± SEM (n = 6).
In conclusion, the results are interesting. In the future could be fascinating to perform this study on lying hens and compare the results.
Response: Thank you for the comment. Your comment has been included in the discussion section as a consideration for future research work (P12 L400).
Comments on the Quality of English Language
The paper is well written, and authors should check for minor misspellings or errors.
Response: Thank you for the comment. The authors have revised the manuscript, and an English editing service has reviewed it for correct grammar and typos.
