Quantitative mRNA and Protein Analyses of ZPB1, ZPB2, and ZPC in Different Genetic Strains of Broiler Breeders

Round 1

Reviewer 1 Report

Quantitative mRNA and protein analyses of ZPB1, ZPB2, and 2 ZPC in different genetic strains of broiler breeder hens.

This study gives us a basic physiological knowledge about the putative relationship between avian ZP proteins and sperm binding/ fertility. Its’ an interesting work and the authors have presented data from multiple experiments that led to this conclusion. This study will be of interest to reproductive physiologists and geneticists. Overall, the manuscript is well-written with good English and well-organized. My specific comments are below.

Comments

Abstract

How is this basic physiological knowledge important from the standpoint of broiler breeder fertility or what is the practical importance of this study?

Introduction

No information on the importance of using the 4 specific genetic lines for comparison is mentioned.

The practical importance of this basic physiological knowledge is not mentioned.

Materials and Methods

Line 89: It is confusing. Did you mean ‘yolk’ instead of ‘follicle’?

Experiments 1 and 2 used birds of different age groups and hence, the direct comparison of results is questionable.

Results

Table1: Be consistent with superscripts chronological order, either use ‘a’ for the highest value or the lowest value for all the parameters in the table.

Discussion

Line 382: What are some examples of such antibodies against ZP glycoproteins or at least the source of such antibodies or are they specifically targeting hepatic-origin ZP proteins? Because a blood-barrier exists between the reproductive system and the body preventing direct contact between antibodies and the ovum. So, it needs to be clarified.

Lines 403-408: Again, the difference in the age of birds used for protein expression and mRNA expression needs to be taken into account while discussing the difference between protein and mRNA expressions.

Author Response

Thank you for taking the time and providing valuable feedback on our submitted manuscript. We know the time and effort that goes into providing constructive criticism. We believe the paper has improved because of your input. We hope that we have sufficiently responded to your constructive criticism. Please let us know otherwise.

Our responses to your prompt are below in red.

 

 

How is this basic physiological knowledge important from the standpoint of broiler breeder fertility or what is the practical importance of this study?

This is a fair criticism concerning the abstract, but difficult to correct due to the word limit for the abstract. The final sentence of the abstract suggests that ZPB2 plays a critical role in sperm binding at the GD region and therefore could be used as a selection tool for increased fertility. Although this is not in the abstract, we suggest the study's practical applications in both the discussion and conclusion.  

 

Introduction

No information on the importance of using the 4 specific genetic lines for comparison are mentioned.

The practical importance of this basic physiological knowledge is not mentioned.

This is an excellent point, and we agree. I have added a new statement to provide more clarity as to why the different lines were used in the study (lines 56-57). Our previous report showed that genetic lines of turkey hens have differential ZP mRNA expression as well as differences in ZP protein abundance in the IPVL. Thus, part of our investigation would determine if different broiler lines that have been subjected to genetic selection in commercial poultry production also differ in their ZP expression. If ZP proteins do change in their expression, it provides evidence that these proteins could be selected for in a selection program for improved fertility.  

Materials and Methods

Line 89: Its confusing. Did you mean ‘yolk’ instead of ‘follicle’?

Thank you for catching this issue, we have made the correction (line 91).

Experiments 1 and 2 used birds of different age groups and hence, the direct comparison of results are questionable.

See the statement below-concerning lines 403-408.

Results

Table1: Be consistent with superscripts chronological order, either use ‘a’ for the highest value or the lowest value for all the parameters in the table.

Thank you for catching that and yes, this was inconsistent. This was corrected in the manuscript with “a” representing the highest to “c” representing the lowest in both Table 1 and Table 2.

 

Discussion

Line 382: What are some examples of such antibodies against ZP glycoproteins or at least the source of such antibodies or are they specifically targeting hepatic-origin ZP proteins? Because a blood-barrier exist between the reproductive system and the body preventing direct contact between antibodies and ovum. So, it needs to be clarified.

We did add a clarification that the antibodies that can lead to loss of ovarian function and depletion of the primordial follicle population must be directed against the common ZP region of the ZP glycoproteins as stated in the referenced papers (line 383).

The ovarian blood-follicle barrier is selectively permeable. For example, IgG, or in the case of the bird IgY, passes into the follicle and is the source of maternal immunity for the chick. Thus, it is expected that exogenous anti-ZP IgG antibodies, which have a lower molecular weight than avian IgY, would retain this capacity and could lead to follicular atresia in small follicles.

Lines 403-408: Again, the difference in the age of birds used for protein expression and mRNA expression needs to be taken in account while discussing the difference between protein and mRNA expressions.

This could be a factor and I have added a statement in lines 403 to 406 to address this concern. It should be noted that this difference agrees with our previously reported turkey data where there was a disagreement between protein abundance in the IPVL and mRNA expression for the same ZP proteins. Note that these differences, between mRNA and protein, were from turkeys of the same age. Although it doesn't mean that this could be a factor, I could not find any reports showing any change in ZP expression associated with age in birds (nor mammals).

Reviewer 2 Report

Manuscript poultry-1849004 entitled “Quantitative mRNA and protein analyses of ZPB1, ZPB2, and ZPC in different genetic strains of broiler breeder hens”. Please notice the following:

General view: The manuscript expressed a novel and great concept in good language and grammar. The manuscript was expressed properly. A few suggestions were provided to improve the expression of the text.

The manuscript could be accepted for publication after minor revision.

Title: Prefered to be more concise as follows: “Quantitative mRNA and protein analyses of ZPB1, ZPB2, and ZPC in different genetic strains of broilers”.  

Abstract: Clear, indicative, and informative.

Introduction: Informative to a high degree, but preferred to rearrange inro three paragraphs only i.e., 1. Introduction 2. Significance of the study, and 3. Aim of the study.

The aim: The aim of the study has to be listed clearly by the end of the introduction triad.

Materials and Methods: Please notice the following:

1.      Please illustrate details on the housing microclimatic conditions including cages design, feeding and watering system, ventilation system, lighting color & intensity, fly control, rodent control, waste management, vaccination act, cleaning protocol, disinfection regimen, and disposal strategies in each of the five experiments.

2.      Specify the total number of granulosa and liver samples.

3.      Specify the total number of Ross 708 broiler breeder pullets in experiment 3.

4.      Specify the total number of Cobb 500 broilers in experiments 4 and 5.

5.      Please provide the statistical model.

Results: Novel, clear, and informative.

Discussion: Properly expressed and contribute to knowledge with a good level of speculations and a high level of comparison.

Conclusion: Clear and informative with a couple of suggestions to improve the expression of the text.

Authors’ contributions: Clear and informative.

Acknowledgment: Not listed.

Funding: Clear and informative.

References: MUST BE UPDATED as only 15.5% (7 out of 45) were published in the past five years. This percentage has to be increased to at least 30-40%.

Tables: Well organized and presented.

Figures: Well organized and presented.

Attached are the manuscript files with all the suggested modifications as pinned comments.

Comments for author File: Comments.pdf

Author Response

Thank you for taking the time and providing valuable feedback on our submitted manuscript. We know the time and effort that goes into providing constructive criticism. We believe the paper has improved because of your input. We hope that we have sufficiently responded to your constructive criticism. Please let us know otherwise.

Our responses to your prompt are below in red.

General view: The manuscript expressed a novel and great concept in good language and grammar. The manuscript was expressed properly. A few suggestions were provided to improve the expression of the text.

The manuscript could be accepted for publication after minor revision.

Thank you.

Title: Prefered to be more concise as follows: “Quantitative mRNA and protein analyses of ZPB1, ZPB2, and ZPC in different genetic strains of broilers”. 

Thank you for the suggestion, we agree and have amended the title.  

Abstract: Clear, indicative, and informative.

Thank you

Introduction: Informative to a high degree, but preferred to rearrange inro three paragraphs only i.e., 1. Introduction 2. Significance of the study, and 3. Aim of the study.

The aim: The aim of the study has to be listed clearly by the end of the introduction triad.

We believe by including a statement (lines 56-67) to address a concern from another reviewer that the aim now has more clarity.

 

Materials and Methods: Please notice the following:

1. Please illustrate details on the housing microclimatic conditions including cages design, feeding and watering system, ventilation system, lighting color & intensity, fly control, rodent control, waste management, vaccination act, cleaning protocol, disinfection regimen, and disposal strategies in each of the five experiments.

In an interest to keep the paper concise, we did not include the above information. We believe the provided information is sufficient for the scope and goal of the study. However, it should be noted that all birds in experiments 3, 4, and 5 were raised using standard conditions at the University of Georgia Poultry Research Center. As stated, the birds were provided a standard broiler breeder pullet diet on a skip-a-day feeding and raised according to the recommended primary breeder guidelines. Also, as stated, both the lighting schedule and feeding regime during production were based on guidelines from the primary breeder. For experiment 1, recommended guidelines from the primary breeder were followed at the University of Arkansas (and previously described in Bakst et al.).

2. Specify the total number of granulosa and liver samples.

I added (n = 26) for liver samples in line 107 to provide more clarity. The n =13 was already present for granulosa samples in line 109. I hope this is satisfactory to your request. 

3. Specify the total number of Ross 708 broiler breeder pullets in experiment 3.

Over 2,000 pullets were used in both the Ross 708 (experiment 3) and Cobb 500 (experiments 4 and 5) experiments. These birds were raised for separate experiments; however, we were able to get samples from the control or standard hens from the experiments for our study. We didn’t want to confuse the reader by having mentioned 2,000 hens when our n was never more than 26 (or 12 for these experiments).  As mentioned in the Methods, 12 were used in experiment 3, and 3 for both experiments 4 and 5. Nevertheless, we would add this should you think it is still needed.

4. Specify the total number of Cobb 500 broilers in experiments 4 and 5.

See above

5. Please provide the statistical model.

Since this experiment was not arranged as a factorial experiment, we believe all of the correct statistical methodologies are provided.

Results: Novel, clear, and informative. 

Thank you

Discussion: Properly expressed and contribute to knowledge with a good level of speculations and a high level of comparison. 

Thank you

 

Conclusion: Clear and informative with a couple of suggestions to improve the expression of the text.

Thank you, we updated the conclusion.

 

 

References: MUST BE UPDATED as only 15.5% (7 out of 45) were published in the past five years. This percentage has to be increased to at least 30-40%.

Agree that most of the referenced papers are out of the suggested range. However, only two labs (ours and one in Japan), actively conduct research on avian ZP proteins. To our knowledge, every avian ZP paper that is relevant to this study and that occurs within the suggested timeframe has been cited. There are several ZP studies in mammalian models that have been completed within this timeframe, but we don’t feel that most were relevant to our study in the avian model.  

 

Thank you again for your valuable input. 

Reviewer 3 Report

The paper contains 5 experiments and investigated an interesting topic fitting the scope of the journal (poultry).

The Authors' findings have been well discussed.

The manuscript has been properly organized and the Tables and figures are clear.

However, some changes may add further value to the paper, such as:

Line 39: other reports….where are these reports? Add some relevant references

Add the hypothesis of the study at the end of the introduction section – before your objectives

Line 409: correct this sentence "Differences in relative abundance of ZP proteins among genetic lines was reported"

In conclusion: more improvements are needed in this section.

Line 465: were not was "Differences in ZPC protein abundance were detected"

There are some linguistic errors

  

Author Response

Thank you for taking the time and providing valuable feedback on our submitted manuscript. We know the time and effort that goes into providing constructive criticism. We believe the paper has improved because of your input. We hope that we have sufficiently responded to your constructive criticism. Please let us know otherwise.

Our responses to your prompt are below in red.

 

The paper contains 5 experiments and investigated an interesting topic fitting the scope of the journal (poultry).

The Authors' findings have been well discussed.

The manuscript has been properly organized and the Tables and figures are clear.

However, some changes may add further value to the paper, such as:

Line 39: other reports….where are these reports? Add some relevant references

Agree that the wording of this statement is confusing. I have substituted “other” for “most” in the sentence to provide more clarity. The purpose of the statement was to prepare the reader for understanding that many papers use the numerical nomenclature system, but we are using the Spargo and Hope nomenclature since we have already published multiple papers using this system.

Add the hypothesis of the study at the end of the introduction section – before your objectives

It is possible that in making a correction from another reviewer, I have addressed this with a lead-in sentence to the purpose of the experiment (lines 56-60).

Line 409: correct this sentence "Differences in relative abundance of ZP proteins among genetic lines was reported"

Thank you for catching that error. It has been fixed.

In conclusion: more improvements are needed in this section.

This is a well-received criticism that was amended. The conclusion was significantly revised to be more concise and draw out the important conclusions.  

Line 465: were not was "Differences in ZPC protein abundance were detected"

Thank you for catching that error. It has been fixed.

There are some linguistic errors

Hopefully, those have been addressed with subsequent corrections.

Thank you for your valuable input on the manuscript. 

Reviewer 4 Report

The manuscript entitled "Quantitative mRNA and protein analyses of ZPB1, ZPB2, and 2 ZPC in different genetic strains of broiler breeder hens" by Benson et al., the results provide further evidence that ZPB2 maybe critical for avian 22 sperm binding at the GD region of the IPVL, and the preparation of this manuscript is also well.

Comments:

1. kindly provide the blots/gels figures for the proteins expression

2. Title: Using the full name of the genes

3. Abstract: Explain abbreviations when they first appear

4. Streamline the conclusion section to present the most important conclusions

Author Response

Thank you for taking the time and providing valuable feedback on our submitted manuscript. We know the time and effort that goes into providing constructive criticism. We believe the paper has improved because of your input. We hope that we have sufficiently responded to your constructive criticism. Please let us know otherwise.

Our responses to your prompt are below in red.

 

 

Comments:

1. kindly provide the blots/gels figures for the proteins expression

We initially had representative blots for the different experiments. However, this significantly lengthened the manuscript and they were cut on the basis that our previous (and referenced) study has representative blots using these same antibodies (reference 17). However, we do not disagree with the statement and would be open to suggestions as to which representative blots should be provided.

2. Title: Using the full name of the genes

Fair point, but all referenced papers, as well as our previous submissions, use the abbreviation ZP(X) for the name. If changed then the title would be extremely long.

3. Abstract: Explain abbreviations when they first appear

Thank you, but both “RT-PCR” and “RNA” are considered common abbreviations that do not need to be defined as stated by MDPI. However, we believe that we have explained all other abbreviations correctly. 

4. Streamline the conclusion section to present the most important conclusions

This is a well-received criticism that was amended. The conclusion was significantly revised to be more concise and draw out the important conclusions.