Integrated Analysis of Testicular Histology, Sperm Quality, and Gene Expression (TGFB2, DMRT1) in Rooster Semen (Gallus gallus domesticus)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Animals 4038852: Integrated analysis of testicular histology, sperm quality, and gene expression (TGFB2, DMRT1) in roosters semen (Gallus Gallus domesticus) .

The production of fertile spermatozoa relies on normal germ cell mitosis and meiosis, as well as the proper functioning of both germ cells and Sertoli cells. Spermatozoa are the final product of the seminiferous tubules. Since the quality of ejaculated sperm reflects the condition of their precursor cells, the morphology and biometry of the seminiferous tubules and their constituent cells are frequently examined.

Firstly, I would like to commend the quality of language used throughout the manuscript. The entire document flows logically. It is straightforward to read and understand. However, there is some inconsistency in the level of detail provided in different sections. In my opinion, some parts of the text are too vague.

SIMPLE SUMMARY

Lines 12-14 should be moved from this section to the abstract. They should replace the first two sentences of the abstract.

The part starting from line 17 and ending at line 24 – should be rewritten as “TGFB2 gene expression in native semen showed no significant association with the studied parameters”. The main dependencies should be listed (in relation to DMRT1 gene expression).

I am uncertain whether to repeat the phrase “native semen” throughout the entire text. In my opinion, this is unnecessary. The fact that the semen comes from a native, conservative breed has already been emphasised in the Materials and Methods section. If you want to emphasise it more, place it in the manuscript title.

ABSTRACT

In my opinion, the abstract should be revised. It is too long-winded, including digressions that are suitable for discussion. The observations should be moved to another section.

INTRODUCTION

This comment applies to the entire text. Not “seed” but “semen”. The word “seed” is usually used in relation to the plant kingdom. You can also use the term “ejaculate”.

Generally, the introduction is engaging and provides a comprehensive explanation of the role of seminiferous tubules and the genes studied in enhancing ejaculate quality in birds.

MATERIALS AND METHODS

In my opinion, this section lacks the most information. All additional information should be completed. To start with:

1. What is the abbreviation described in GOST 27267-87 RU? This should be explained in the text.
2. Measuring concentration with a photometer is not entirely suitable, as this device also counts all somatic cells in the final result.
3. Why did you extract the right testicle for testing in the methodology provided, even though in birds the left one is larger and better developed?
4. What exactly was the process of isolating RNA from spermatozoa? As far as I know, this process is not straightforward; I would describe it as very demanding. Low RNA content, high DNA content, RNase activity, and the presence of lipids and proteins can seriously interfere with it. Therefore, a detailed step-by-step protocol for RNA isolation (including specific centrifugation steps, the use of detergents if necessary, removal of somatic cells, etc.) should be provided. I also have a question regarding the amount of RNA recovered: was there enough for all analyses?
5. Did you use any classical cell marker genes for somatic cells to verify their absence in the biological material?

RESULTS

Line  240 - I would expand abbreviations as they appear in the text for the first time.

What are the “units” given in lines 275-277?

There is an error in Table 3 – the lack of a ± symbol in one value.

The abbreviation “pcs.” in line 286 – what does it mean?

How to explain the relationship between the total cross-sectional area of the seminiferous tubules and the sperm viability? This is the only dependency that has not been described.

For me, the gene expression was too variable to draw any accurate conclusions – maybe you should mention in the title that the results are from preliminary studies? In the future, I would expand the experiment to include a larger number of individuals. I know that this is a conservative breed, but perhaps it would be possible to increase the size of the study group next time?

You mentioned that the reproductive status of birds might have varied. Don't you think it could have been caused by the age of the individuals used in the experiment? You have given the age range of 28-32 weeks. That is quite a long time for roosters. Perhaps it would be worth reducing the range to two weeks in the future?

Lines 389-391 - What does this description belong to?

DISCUSSION

I find the discussion to be relatively light, coherent, and accessible. The references cited here further elaborate on the morphometric dependence of seminiferous tubules on sperm quality, as well as on the expression patterns of DMRT1 and TGFB2. The results concerning the expression of DMRT1 are of particular interest.

CONCLUSIONS

Conclusions are formulated in a comprehensible manner. However, I would expand on the sentence “It was also found that high expression of the DMRT1 gene (where?) may be a marker of immaturity of the spermatogenic process, while the TGFB2 gene performs background regulatory functions (by what?) at the molecular level under non-selective conditions”.

REFERENCES

The references section lists 51 original scientific papers, the most recent and directly relevant in describing the morphometry of reproductive organs and their association with spermatogenesis. This suggests that the authors are up to date in the field.

IN SUMMARY

In my view, this manuscript showcases good scientific writing. The subject under investigation remains poorly understood, which enhances its importance. Although the manuscript has certain limitations that should be addressed, it still has the potential to provide novel insights into the rooster’s reproductive tract physiology.

Given the current content and structure, I recommend substantial revisions to improve clarity, focus, and thoroughness.

 

Author Response

Dear reviewers, thank you for your careful and kind review of this article. Your comments and advice are greatly appreciated. We have endeavored to fully address all comments and have made corrections to the manuscript.

SIMPLE SUMMARY

Lines 12-14 should be moved from this section to the abstract. They should replace the first two sentences of the abstract.

Reply: We rewrote the abstract.

Reply: corrected

The part starting from line 17 and ending at line 24 – should be rewritten as “TGFB2 gene expression in native semen showed no significant association with the studied parameters”. The main dependencies should be listed (in relation to DMRT1 gene expression).

Reply: We rewrote the abstract.

I am uncertain whether to repeat the phrase “native semen” throughout the entire text. In my opinion, this is unnecessary. The fact that the semen comes from a native, conservative breed has already been emphasised in the Materials and Methods section. If you want to emphasise it more, place it in the manuscript title.

Reply: When writing this article, the term "native" was used to refer to fresh, untreated ejaculate; for clarity, it has been replaced with "fresh" throughout the text.

In my opinion, the abstract should be revised. It is too long-winded, including digressions that are suitable for discussion. The observations should be moved to another section.

INTRODUCTION

This comment applies to the entire text. Not “seed” but “semen”. The word “seed” is usually used in relation to the plant kingdom. You can also use the term “ejaculate”.

Reply: Thank you for your comment; we have corrected the technical errors in the manuscript text.

Generally, the introduction is engaging and provides a comprehensive explanation of the role of seminiferous tubules and the genes studied in enhancing ejaculate quality in birds.

Reply: Thank you for your appreciation of the presented research.

MATERIALS AND METHODS

In my opinion, this section lacks the most information. All additional information should be completed. To start with:

1. What is the abbreviation described in GOST 27267-87 RU? This should be explained in the text.

Reply: Added transcript to text: Russian: State Standard

 

1. Measuring concentration with a photometer is not entirely suitable, as this device also counts all somatic cells in the final result.

Reply: The Accuread Photometer, used to determine the concentration of sperm in the ejaculate, is positioned by the manufacturer specifically as a device for determining the concentration of sperm (https://www.imv-technologies.us/product/poulrty-accuread-photometer)

 

1. Why did you extract the right testicle for testing in the methodology provided, even though in birds the left one is larger and better developed?

Reply: In this case, the choice of the left or right testicle was not considered significant, since, in our opinion, this is not fundamental for the histological analysis

 

1. What exactly was the process of isolating RNA from spermatozoa? As far as I know, this process is not straightforward; I would describe it as very demanding. Low RNA content, high DNA content, RNase activity, and the presence of lipids and proteins can seriously interfere with it. Therefore, a detailed step-by-step protocol for RNA isolation (including specific centrifugation steps, the use of detergents if necessary, removal of somatic cells, etc.) should be provided. I also have a question regarding the amount of RNA recovered: was there enough for all analyses?

 

 

Reply: We have supplemented the Materials and Methods section, item 2.3. RNA isolation, by adding a detailed step-by-step protocol.

 

1. Did you use any classical cell marker genes for somatic cells to verify their absence in the biological material?

 

Reply: Thank you for your recommendation. In our study, we did not use somatic cell marker genes (e.g., VIM (vimentin) for fibroblasts and macrophages, CD45 (for leukocytes), and KRT (keratins) for epithelial cells) to confirm their absence in the semen samples. However, as stated in the methodology, all semen samples were subjected to mandatory pre-clearance of somatic cells before RNA extraction. This approach minimized RNA contamination with somatic cells, as evidenced by:

- High RNA purity (A260/A280 = 1.9–2.1; A260/A230 > 2.0);

- Expectedly low expression levels of genes active in the early stages of spermatogenesis (e.g., DMRT1 in mature sperm), consistent with the literature;

- No signs of degradation or abnormal mRNA profiles, typical of mixed cell populations.

 

RESULTS

Line  240 - I would expand abbreviations as they appear in the text for the first time.

Reply: transcripts were given in the section "Materials and Methods" line 173

What are the “units” given in lines 275-277?

Reply: changed to “cells”

There is an error in Table 3 – the lack of a ± symbol in one value.

Reply: added

The abbreviation “pcs.” in line 286 – what does it mean?

Reply: “pcs.” - meant the number of pieces; for convenience and clarity, the headings in Table 3 were changed to “Number of…..”

How to explain the relationship between the total cross-sectional area of the seminiferous tubules and the sperm viability? This is the only dependency that has not been described.

Reply: We agree with your comment. We, too, were surprised by this correlation. We present our hypotheses regarding this relationship between viability and the cross-sectional area of ​​the seminiferous tubules, as well as other parameters, in the "Discussion" section, starting at line 418.

For me, the gene expression was too variable to draw any accurate conclusions – maybe you should mention in the title that the results are from preliminary studies? In the future, I would expand the experiment to include a larger number of individuals. I know that this is a conservative breed, but perhaps it would be possible to increase the size of the study group next time?

Reply: Thank you very much for your advice, we agree with your comment. We have a series of studies planned on this topic to ensure repeatability of the results.

You mentioned that the reproductive status of birds might have varied. Don't you think it could have been caused by the age of the individuals used in the experiment? You have given the age range of 28-32 weeks. That is quite a long time for roosters. Perhaps it would be worth reducing the range to two weeks in the future?

Lines 389-391 - What does this description belong to?

Reply: Thank you for your comment; we have corrected the technical errors in the manuscript text.

 

CONCLUSIONS

Conclusions are formulated in a comprehensible manner. However, I would expand on the sentence “It was also found that high expression of the DMRT1 gene (where?) may be a marker of immaturity of the spermatogenic process, while the TGFB2 gene performs background regulatory functions (by what?) at the molecular level under non-selective conditions”.

Reply: thanks for the clarification, we have made an adjustment.

"It was also found that high expression of the DMRT1 gene in rooster semen may be a marker of immaturity of the spermatogenic process, while the TGFB2 gene performs background regulatory functions (the regulation of spermatogenesis and testicles de-velopment) at the molecular level under non-selective conditions."

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

To Authors,

This manuscript presents an integrated of male reproductive function in roosters by combining testicular histology, semen quality assessment, and molecular gene expression analysis. The study addresses an important topic in poultry reproductive biology, particularly the need to link structural, functional, and molecular indicators of sperm quality in breeding males. The experimental design is coherent and well-aligned with the stated objectives, and the methodological approaches (histomorphometry, CASA-based semen evaluation, and qPCR-based gene expression) are appropriate and current. However, there are some questions need to be completed as follows.

Major comments:

1. Can DMRT1 expression in native semen be validated as a reliable biomarker for predicting fertility outcomes (e.g., fertilization rate or hatchability) in artificial insemination programs? Please add more information.
2. How does the expression of DMRT1 and TGFB2 in semen compare with their expression levels directly within testicular tissue across different stages of sexual maturity? Please add more information and related references.
3. In discussion section, would the observed correlations between histomorphometric traits and semen quality remain consistent in larger populations or across different chicken breeds (commercial vs. native)? If yes, please compared this results and previous study.
4. How do environmental factors such as heat stress, nutrition, or housing system influence the relationships between testicular morphology, gene expression, and semen quality? Because the researchers did not mention the controlled environmental factors in this study.
5. Are other spermatogenesis-related genes (e.g., SOX9, DAZL, STRA8) more sensitive or complementary markers to DMRT1 when assessing sperm functional maturity?
6. Can integrating molecular markers with conventional semen evaluation improve the accuracy of selecting superior roosters for long-term genetic improvement programs? Please add more information.

Minor comment:

1. Line 3: In the title, “roosters semen” should be revised to “rooster semen”.
2. Gene symbols (e.g., DMRT1, TGFB2) should be consistently italicized throughout the text, figures, and tables, following standard genetic nomenclature.
3. Ensure consistent capitalization (avoid mixing DMRT1, or Dmrt1).
4. Several journal titles in the reference list are inconsistently abbreviated (e.g., world’s poult. sci., Biol. of reprod., Plos one). Please standardize all journal names according to MDPI or PubMed journal abbreviation style (e.g., World’s Poult. Sci. J., Biol. Reprod., PLOS ONE).
5. Units should be consistently formatted with a space between number and unit (e.g., “305.4 microns” → 4 µm). Use standard SI units and symbols (µm instead of microns) throughout the manuscript.
6. Decimal separators alternate between commas and periods (e.g., 0,651 vs. 0.651). Please standardize to decimal points throughout all tables and text, as required by international journals.
7. When reporting correlation coefficients, use consistent notation (e.g., r = −0.782, p < 0.05). Clarify in the Methods whether the reported threshold |r| ≥ 0.648 was predefined or derived from sample size considerations.
8. The phrase “Informed consent was obtained from all subjects involved in the study” is not fully appropriate for animal studies and may be revised to reflect animal-use ethics approval only.
9. The number of keywords list are relatively long; consider reducing redundancy and prioritizing searchable terms (e.g., “morphometry,” “sperm quality,” “rooster”). Keywords: Three to ten pertinent keywords need to be added after the abstract. We recommend that the keywords are specific to the article, yet reasonably common within the subject discipline.

Author Response

Dear reviewers, thank you for reviewing our manuscript.

Your comments have undoubtedly improved the quality of the submitted material.

We have endeavored to respond to all comments in as much detail as possible for your convenience.

 

Major comments:

1. Can DMRT1 expression in native semen be validated as a reliable biomarker for predicting fertility outcomes (e.g., fertilization rate or hatchability) in artificial insemination programs? Please add more information.

Reply: In our opinion, this is a reliable marker for assessing semen motility and viability, but there are simpler and less expensive methods for predicting fertility.

 

1. How does the expression of DMRT1 and TGFB2 in semen compare with their expression levels directly within testicular tissue across different stages of sexual maturity? Please add more information and related references.

Reply: Thank you very much for the advice, this was not part of the experimental design, but we plan to study the expression of DMRT1 and TGFB2 in testicular tissue in our next studies.

 

1. In discussion section, would the observed correlations between histomorphometric traits and semen quality remain consistent in larger populations or across different chicken breeds (commercial vs. native)? If yes, please compared this results and previous study.

Reply: Thank you for your question. We've covered this in detail in the Discussion section, which includes data on commercial meat and egg chicken breeds, the Chinese aboriginal breed, and various mammal species.

 

1. How do environmental factors such as heat stress, nutrition, or housing system influence the relationships between testicular morphology, gene expression, and semen quality? Because the researchers did not mention the controlled environmental factors in this study.

Reply: The experimental rooster flock was kept in stable conditions, maintaining a stable microclimate, lighting regime, and water and feeding schedules. Therefore, we did not consider environmental factors influencing the parameters studied.

 

1. Are other spermatogenesis-related genes (e.g., SOX9, DAZL, STRA8) more sensitive or complementary markers to DMRT1 when assessing sperm functional maturity?

 

Reply: The DMRT1 gene is an evolutionarily conserved transcription factor central to the initiation and maintenance of spermatogenesis in birds, including chickens (Gallus gallus). Our analysis revealed an inverse correlation between DMRT1 expression levels in sperm and indicators of their functional maturity, such as viability and motility. This suggests that high levels of residual DMRT1 mRNA in mature sperm may reflect incomplete spermiogenesis, where the "immature" transcriptome has not been fully degraded.

DOI: 10.1038/nature08298

As for other genes associated with spermatogenesis, such as SOX9, DAZL, and STRA8, their important functions have indeed been described in the scientific literature:

• SOX9 is involved in early Sertoli cell differentiation and seminiferous tubule formation;
• DAZL is a key regulator of meiosis and mRNA translation in germ cells;
• STRA8 is required for the initiation of meiosis in male mammals.

However, in birds, the role of these genes has significant differences:

• SOX9, unlike in mammals, is not a key determinant of male development in birds; its expression is not restricted to Sertoli cells and can vary depending on the developmental stage.
• STRA8 in birds is not found in the chicken (Gallus gallus) genome, as confirmed by Ensembl data and the work of Smith et al. (2009), making it inapplicable as a marker for this species. Ensembl Genome Browser (GRCg6a/Gallus_gallus-5.0),
• DAZL, although present, is expressed predominantly during meiosis and early spermatogenesis, and its residual mRNA in mature sperm, like DMRT1, may serve as a marker of incomplete differentiation. However, in our study, we did not include DAZL in the gene panel, as our initial hypothesis focused on investigating the regulatory networks associated with DMRT1 and TGF-β. DOI: 10.7150/ijbs.11536
•  

Thus, the DMRT1 gene currently remains the most specific and sensitive marker for assessing sperm functional maturity in roosters, as it is directly linked to the regulation of spermatogenesis at the transcriptional level, and its expression in mature gametes has clear prognostic value. The SOX9 and STRA8 genes are not relevant markers for birds, and DAZL, despite its potential usefulness, requires a separate validation study. We plan to include it in future studies for a comprehensive assessment of sperm molecular maturity.

 

 

1. Can integrating molecular markers with conventional semen evaluation improve the accuracy of selecting superior roosters for long-term genetic improvement programs? Please add more information.

Reply: In our opinion, before introducing molecular markers associated with the reproductive function of roosters into genomic selection, it is necessary to conduct more large-scale studies on industrial chicken populations.

Minor comment:

1. Line 3: In the title, “roosters semen” should be revised to “rooster semen”.

Reply: corrected

 

1. Gene symbols (e.g., DMRT1, TGFB2) should be consistently italicized throughout the text, figures, and tables, following standard genetic nomenclature.

Reply: corrected

 

1. Ensure consistent capitalization (avoid mixing DMRT1, or Dmrt1).

Reply: In this case, this is the original title of the article by other authors, we provide a link to the source https://doi.org/10.1093/jeb/voaf031

 

1. Several journal titles in the reference list are inconsistently abbreviated (e.g., world’s poult. sci., Biol. of reprod., Plos one). Please standardize all journal names according to MDPI or PubMed journal abbreviation style (e.g., World’s Poult. Sci. J., Biol. Reprod., PLOS ONE).

Reply: Fixed to comply with MDPI requirements

 

 

1. Units should be consistently formatted with a space between number and unit (e.g., “305.4 microns” → 4 µm). Use standard SI units and symbols (µm instead of microns) throughout the manuscript.

Reply: corrected

 

1. Decimal separators alternate between commas and periods (e.g., 0,651 vs. 0.651). Please standardize to decimal points throughout all tables and text, as required by international journals.

Reply: corrected

 

1. When reporting correlation coefficients, use consistent notation (e.g., r = −0.782, p < 0.05). Clarify in the Methods whether the reported threshold |r| ≥ 0.648 was predefined or derived from sample size considerations.

 

Reply: In our study, we used the Spearman’s rank correlation coefficient (r_s) to assess monotonic relationships between variables with a sample size of n = 10.

To determine the statistical significance of correlations, we used a significance level of α = 0.05. With this sample size, the critical value of Spearman's rank correlation coefficient is |r_s| = 0.648 (two-tailed test). This means that any correlation with |r_s| ≥ 0.648 will have a p < 0.05 and is therefore considered statistically significant.

In the text of the article, we indicated:

 

|r_s| ≥ 0.648 — to clearly define the significance threshold in terms of the correlation coefficient itself, which is convenient for interpreting heat maps and tables;

p < 0.05 — as the generally accepted level of statistical significance.

Thus, both notations refer to the same significance criterion, simply expressed on different scales:

0.648 is the critical value r_s,

0.05 is the corresponding p-value

 

1. The phrase “Informed consent was obtained from all subjects involved in the study” is not fully appropriate for animal studies and may be revised to reflect animal-use ethics approval only.

Reply: Thanks for the comment, we've removed the item.

 

1. The number of keywords list are relatively long; consider reducing redundancy and prioritizing searchable terms (e.g., “morphometry,” “sperm quality,” “rooster”). Keywords: Three to ten pertinent keywords need to be added after the abstract. We recommend that the keywords are specific to the article, yet reasonably common within the subject discipline.

            Reply: We agree with your comment, we have reduced the number of keywords.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This study identified correlations between the histomorphological structure of the testicles, the parameters of native semen, and the expression level of key spermatogenesis genes – TGFB2 and DMRT1 in roosters and provided lots of basic data. Overall, this manuscript is well written, however, several issues should be revised before publishing.

1. The introduction section is too long.
2. "Figure 5. Expression levels of DMRT1 and TGFB2 genes in native rooster semen" should be divided into two figures, one is for DMRT1 gene and the other for TGFB2 gene. The expression levels should list some other different tissues for comparisons. 

Author Response

Dear reviewer, thank you for reading our manuscript. We have done our best to address all your comments. With gratitude, the authors.

 

1. The introduction section is too long.

 

Reply: Thank you for your comments, but we have tried to cover as much of the most recent and directly related information as possible regarding the morphometry of reproductive organs and their relationship to spermatogenesis, as well as the expression levels of key spermatogonial genes.

However, we have reduced the length of the Simple Summary and Abstract sections.

 

 

1. "Figure 5. Expression levels of DMRT1 and TGFB2 genes in native rooster semen" should be divided into two figures, one is for DMRT1 gene and the other for TGFB2 gene. The expression levels should list some other different tissues for comparisons. 

 

Reply: We agree that separating the graphs can be useful in some cases; however, in this case, we deliberately left the expression of the TGFB2 and DMRT1 genes on a single graph for the following reasons:

In our opinion, this arrangement of the expression diagram of both genes in Figure 5 makes the material easier to understand.

1. Comparative clarity: The combined graph allows the reader to assess at a glance the differences in the distribution and expression levels of two key genes. This is especially important, as both genes play a fundamental role in the regulation of spermatogenesis, and their comparison is one of the key findings of the study.
2. Single y-axis scale: Using a single scale for both genes (0–4.0) ensures a clear visual comparison of expression levels, which would be impossible if the graphs were separated using different scales.
3. Space saving and article structure: Separating the graphs would have resulted in larger figures and duplicated legends, which contradicts the principle of compactness and clarity adopted in the journal Animals (MDPI). Most molecular biology publications that compare two or more genes use a combined boxplot (e.g., [Guo et al., 2024; Zhang et al., 2023]).

Therefore, we believe that the current data presentation format is optimal for conveying the scientific message and does not require modification.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

First, I would like to thank the authors for their detailed responses, which have satisfactorily addressed my main concerns. Nevertheless, I remain convinced that the left and right testicles may differ in size, depending of course on the reproductive season and individual characteristics, and that the histological dimensions of individual regions of the seminiferous tubules may therefore also vary. If further research is planned, it may be worthwhile to include the left testicle in future examinations as well.

Regarding the photometer and concentration measurements, I am not entirely convinced, although I trust the experience of the authors. My own laboratory experience differs, and in other animal species such measurements are not always fully accurate.

In summary, I carefully checked the revised manuscript once again. The chapters that required correction now show clear improvement, with stronger organisation and a more consistent academic tone. The abstract is better constructed, with clearer emphasis on the methodology, and the methodological chapters themselves are better structured and clarified. The analysis chapters are also more coherent and better supported.

Overall, the revisions have significantly strengthened the quality, clarity, and readability of the manuscript. In my opinion, the manuscript has been substantially improved and can be published in the journal in its current form.

Author Response

Dear reviewers, we thank you for your high praise of our work.
We wish you a Happy New Year!
Sincerely, the team of authors

Reviewer 2 Report

Comments and Suggestions for Authors

Accept in present form

Author Response

Dear reviewers, we have made the necessary changes to the manuscript to improve the English language.
Sincerely, the authors

Reviewer 3 Report

Comments and Suggestions for Authors

As my 2nd question "Figure 5. Expression levels of DMRT1 and TGFB2 genes in native rooster semen" should be divided into two figures, one is for DMRT1 gene and the other for TGFB2 gene. The expression levels should list some other different tissues for comparisons." 

The author may not really understood. I means the gene expression comparing should do between different tissues. I suggested the authors at least should explain this issue in the discussion section.

Others are OK.

Author Response

Dear Reviewer,

Thank you very much for your constructive comments and valuable suggestions. We have carefully revised the manuscript in accordance with your feedback and believe that the changes made will significantly enhance the clarity and scientific rigor of our work.

In response to your comment, we have completely revised this part of the manuscript to divide Figure 5 and provide a comparison of gene expression in different tissues in section the Discussion. The original composite drawing has now been divided into two separate parts to improve readability and biological interpretation. In particular, Figure 5 -  shows the relative expression of DMRT1 in rooster sperm (mean ± SD, n = 10), while Figure 6 -  shows the same for TGFB2, both based on our experimental PCR data.

You can see the changes made:

 in the Materials and Methods section  lines 225-248

in the Results section lines 388-393

 in the Discussion section, lines 515-531, 560-569.

The additions to the Discussion section were based on a comparison of our results with Bgee data (https://www.bgee.org/bgee15-2/search/genes ). Figures demonstrating our reasoning are presented below.

We decided not to include them in the manuscript, as they would complicate its understanding, and limited ourselves to the discussion.

Figure 5С. Comparative Analysis of DMRT1 Expression: qPCR Data in Sperm vs RNA-seq Scores from Bgee Database

Note: Bgee scores are based on RNA-seq data and represent expression relative to the genome-wide average (scale 0–100). RQ values are from qPCR experiments in sperm, normalized to GAPDH in kidney. Direct statistical comparison is not feasible due to different normalization methods.

Figure 5D. TGFB2 Expression in Rooster Spermatozoa (This Study) vs Public RNA-seq Data from Bgee (Selected Tissues)

Note: Expression scores for selected anatomical units are from the Bgee database (v15+, RNA-seq data), normalized to a 0–100 scale relative to the genome-wide average. Relative expression in sperm (RQ) was measured by qPCR and normalized to GAPDH in kidney tissue. Direct statistical comparison is not feasible due to differing normalization methods. The tissues shown were selected to represent high, medium, and low expression levels for biological context.

 

Thank you again for your valuable comments, and we hope our responses and additions were sufficiently comprehensive.

Sincerely, the team of authors

Author Response File: Author Response.docx