Sex Determination, Evolution of Gonadal Stage in Females, and Seasonal Evolution of Sperm Production in Chelon labrosus (Risso, 1827)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

The authors claim that the steroid hormone analysis allowed the sexual determination, but surely only the gonadal biopsy revealed if the fish were female or male. It is known that sexual hormones in fish are very variable and their combination can manifest differently between males and females, especially in different reproductive seasons, as well as in different periods of sexual maturity.

In this regard, it is essential that the manuscript presents a graph with the plasma sex hormones profile found.

Furthermore, gonadal stages must be shown by imaging the cannulated oocytes or by histology of the gonads. Only then the sexual determination can be associated with different plasma hormone levels.

The discussion is very superficial. It should be more robust.

I didn't understand the Fig 1. If 20 females were analyzed, what is the number shown in development of oocyte diameter? The “mean ± SE” is ok! But the numbers “n= 11, n = 9, n = 1….” are the number of fish or oocytes?

In the case of males, I don't know if the authors can really claim that in the males 3 and 4 the loss of motility was compensated by an increase in the sperm density. This sentence is confusing. Please rewrite it.

In view of the above, I suggest that the authors review their results and resubmit the manuscript at another opportunity.

Author Response

We thank you in advance for taking the time to review the manuscript, we are convinced that with the changes included the work will meet your expectations. We present the corrections and/or explanations under your comments in order to facilitate their validation:

The authors claim that the steroid hormone analysis allowed the sexual determination, but surely only the gonadal biopsy revealed if the fish were female or male. It is known that sexual hormones in fish are very variable and their combination can manifest differently between males and females, especially in different reproductive seasons, as well as in different periods of sexual maturity.

In this regard, it is essential that the manuscript presents a graph with the plasma sex hormones profile found. Furthermore, gonadal stages must be shown by imaging the cannulated oocytes or by histology of the gonads. Only then the sexual determination can be associated with different plasma hormone levels.

We appreciate this comment from the reviewer. Indeed, hormone levels can be highly variable over time. We have incorporated a biplot showing the levels of both hormones in all fish sampled, highlighting those where sex identification with the proposed ratio gave erroneous results (L196). According to the need of presenting images of the cannulated egg, this is not a specific work related to females development, include sex determination, and males sperm evolution study, so that in terms of females we only studied diameter as a well identified indicator of female maturity. Moreover we did not use any product to clarify the egg structure such as acetic, or Serra, so that we haven’t used the images obtained in the manuscript, because they do not provide information about the oocytes structure.

The discussion is very superficial. It should be more robust.

As explained above, this work presents practical tools for the reproductive management of the species in culture conditions. This fact may give the false impression that the discussion is superficial, as well as the fact that it does not present details of the ultrastructure of the ovary. Nevertheless, the conclusions obtained are well justified and supported by the scarce specific bibliography of this species and the available bibliography of related species, and they propose advances in the reproductive control of the species in zootechnical terms.

I didn't understand the Fig 1. If 20 females were analyzed, what is the number shown in development of oocyte diameter? The “mean ± SE” is ok! But the numbers “n= 11, n = 9, n = 1….” are the number of fish or oocytes?

Twenty fish were monitored, of which 14 were females (line 138). In Figure ,1 n = x indicates the number of females from which oocytes could be obtained out of a total of 14 females monitored. We have clarified this in the figure caption as follows:

Figure 1. Development of oocyte diameter (mean ± SE) in Chelon labrosus females during the 2023 breeding season. n indicates the number of females from which a sample was obtained by gonadal biopsy.

In the case of males, I don't know if the authors can really claim that in the males 3 and 4 the loss of motility was compensated by an increase in the sperm density. This sentence is confusing. Please rewrite it.

Amended as suggested (L231-233)

Reviewer 2 Report

Comments and Suggestions for Authors

The abstract lacks clarity and fails to adhere to the standards of scientific manuscript writing. It lacks representation of data and fails to present valid conclusions. Additionally, the abstract contains an excessive number of objectives, which is not typical for this section.

Furthermore, the objectives outlined in the abstract are not adequately reflected in the introduction section. Moreover, the introduction does not provide sufficient background information about the current study.

It is recommended that the author includes information on the histo-architecture of the gonadal stage in the species under consideration.

The manuscript lacks a proper design, and the representation of data is inadequate.

The discussion section is deficient in meaningful discussion, with much of the content being superficial.

Comments on the Quality of English Language

The English language usage in the manuscript is suboptimal and requires significant improvement.

Author Response

We thank you in advance for taking the time to review the manuscript, we are convinced that with the changes included the work will meet your expectations. We present the corrections and/or explanations under your comments in order to facilitate their validation:

The abstract lacks clarity and fails to adhere to the standards of scientific manuscript writing. It lacks representation of data and fails to present valid conclusions. Additionally, the abstract contains an excessive number of objectives, which is not typical for this section.

Amended as suggested.

Furthermore, the objectives outlined in the abstract are not adequately reflected in the introduction section. Moreover, the introduction does not provide sufficient background information about the current study.

It is recommended that the author includes information on the histo-architecture of the gonadal stage in the species under consideration.

As explained in the introduction of the work, the objectives are to deepen the knowledge aimed at controlling the reproduction in captivity of the species for the potential development of industrial cultivation of the species. In a basic science perspective it would have made sense to sacrifice animals for the structural study o the gonad, but because we study many other aspects of the reproduction with this species and we have a limited stock, we don´t find animal sacrifice sufficiently justified. Moreover, we studied diameter as a parameter widely used to determine the evolution of maturity in fish, and unfortunately, we did not use any product to clarify the egg structure such as acetic, or Serra, so that we haven’t used the images obtained in the manuscript, because they do not provide information about the oocytes structure. We will take it into account for future research.

The manuscript lacks a proper design, and the representation of data is inadequate.The discussion section is deficient in meaningful discussion, with much of the content being superficial.

The work presented for publication has a fundamentally applied science approach, with a clear objective: to advance in the zootechnical development of a species with potential for sustainable diversification of aquaculture. Other lee zootechnical approaches are of great interes to us, and we consider them for future research, but they have not been the subject of this study, which may give the false impression that the content or the discussion lacks depth due to the lack of some basic science approaches. Nevertheless, we strongly believe that the work include results of high scientific value and broaden the knowledge related the reproductive biology of the species. It also represent a good starting point for the development of future zootechnical research to enrich the indexed scientific literature of Chelon Labrosus aquaculture.

Comments on the Quality of English Language. The English language usage in the manuscript is suboptimal and requires significant improvement.

Amended as suggested.

Reviewer 3 Report

Comments and Suggestions for Authors

Overall comment: due to the treated topic, also considering that the manuscript studied cryptic family, more importance on species identification should considered within the entire manuscript, starting from the introduction section. Indeed, this represents a possible source of bias within this family as reported by many authors:

https://doi.org/10.3390/su14010398

https://doi.org/10.1111/j.1439-0426.2008.01170.x

Moreover, the sexual dimorphism of the treated species was not mentioned at all. Why do the authors propose a sex identification method based on blood analyses and elaborations, ignoring at the same time the most simple (if possible) morphological sex determination? A good relapse of this document could be, in my opinion not only for the aquaculture field but also for zoology, a possible connection crossing these two methods to give some insights about the hard-to-do sex determination by morphology in Mugilidae species. Have the authors given attention to these aspects and could provide more data about this linking their sex determination results with morphological features of the selected specimens?

Keywords: please enrich the keywords avoiding the use of words already reported in the title.

The Mugilidae species have also been used for decades to produce mullet bottarga. Please consider this aspect in the introduction section. Moreover, the last part of the introduction section which introduces the aims of the study could results unclear in the present form. Consider rewriting it as the abstract ones (as a list).

2.1 - Please quantify the captured and retained in the facility fish, in the present form its unclear the number of utilized specimens.

Please revise the discussion and conclusion considering my first overall comment.

 

Best regards 

The reviewer

 

Author Response

We thank you in advance for taking the time to review the manuscript, we are convinced that with the changes included the work will meet your expectations. We present the corrections and/or explanations under your comments in order to facilitate their validation:

Overall comment: due to the treated topic, also considering that the manuscript studied cryptic family, more importance on species identification should considered within the entire manuscript, starting from the introduction section. Indeed, this represents a possible source of bias within this family as reported by many authors:

We fully agree with the reviewer that taxonomic identification of species of the family Mugilidae is somewhat difficult. When we started working with this species, the first thing we did was to learn to identify it and differentiate it from the other species that cohabit our study area. To do so, we relied on specialists in fisheries biology from our institute, and we used the most advanced identification keys available, following external (Chervinski, 1984; Lloris, 2015) and internal morphological characters (Arias, 1990; Arias & Drake, 1990; only in dead specimens).

Moreover, the sexual dimorphism of the treated species was not mentioned at all. Why do the authors propose a sex identification method based on blood analyses and elaborations, ignoring at the same time the most simple (if possible) morphological sex determination?

As indicated in the Introduction section, outside the reproductive season, gonadal samples cannot be obtained as the gonads are retracted and no sample can be obtained by cannulation or abdominal massage. This is why the hormonal method is proposed as an alternative in these cases, as stated in lines 66-68.

A good relapse of this document could be, in my opinion not only for the aquaculture field but also for zoology, a possible connection crossing these two methods to give some insights about the hard-to-do sex determination by morphology in Mugilidae species. Have the authors given attention to these aspects and could provide more data about this linking their sex determination results with morphological features of the selected specimens?

The work is focused on providing useful tools for the management of Chelon labrosus broodstock. We believe that deviating from this topic to zoological issues would be a diversion from the objective of the study. We agree with the reviewer that the results of our work could be useful in a comparative study for sex determination in specimens of the family Mugilidae. However, we are sorry but we believe that this is outside the scope of our study.

Keywords: please enrich the keywords avoiding the use of words already reported in the title.

Amended as suggested. (L29-30)

The Mugilidae species have also been used for decades to produce mullet bottarga. Please consider this aspect in the introduction section. Moreover, the last part of the introduction section which introduces the aims of the study could results unclear in the present form. Consider rewriting it as the abstract ones (as a list).

Thanks for the contribution that has been included in the introduction of the manuscript.

2.1 - Please quantify the captured and retained in the facility fish, in the present form its unclear the number of utilized specimens.

The number of specimens sourced for the creation of our breeding stock is irrelevant to the understanding of the study. The specimens used in each section of the study are clearly mentioned in the corresponding sections of the paper:

2.2. Sex identification: n = 25 (line 111).

2.3. Gonadal development monitoring and sperm analysis: n = 20 (14 females + 6 males) (line 138-139).

Please revise the discussion and conclusion considering my first overall comment.

Considering the overall comment, and welcoming the reviewers comment, that will surely increase the quality if this manuscript. We think that modifying the structure and discussion to give a zoological approach does not fit with the marked zootechnical character of this study. However, we will consider the suggestions for possible future collaboration with other research groups to deepen the knowledge of the aspects highlighted.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript "Sex determination, evolution of gonadal stage in females and seasonal evolution of sperm production in Chelon labrosus (Risso, 1827)."  aims to increase knowledge on the reproduction of the species to achieve breeding control in captivity conditions, and to provide information to assist in the development of protocols for reproductive control.

The manuscript is clear, easy to follow and interesting. 

The methods are adequately described and the results are clearly presented.

I would only like to see a possible explanation for the inhibition of the final oocyte maturation process observed in the study.

Having nothing more to add I wish the authors all the best.

Author Response

We thank you in advance for taking the time to review the manuscript, we are convinced that with the changes included the work will meet your expectations. We present the corrections and/or explanations under your comments in order to facilitate their validation:

The manuscript "Sex determination, evolution of gonadal stage in females and seasonal evolution of sperm production in Chelon labrosus (Risso, 1827)."  aims to increase knowledge on the reproduction of the species to achieve breeding control in captivity conditions, and to provide information to assist in the development of protocols for reproductive control.

The manuscript is clear, easy to follow and interesting. 

The methods are adequately described and the results are clearly presented.

I would only like to see a possible explanation for the inhibition of the final oocyte maturation process observed in the study.

Amended as suggested (L294-296).

Having nothing more to add I wish the authors all the best.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

I understand that the objective of the work is not to showcase the structure of oocytes. However, working with gonadal stages without illustrating the morphology of the gonad seems odd to me, especially since, as you mention, the levels of sexual hormones are highly variable. It is necessary to consider more than one parameter to determine the gonad stages. So, in my opinion, either present some image of the gonad (or oocyte), or you should consider changing the focus of the manuscript. I leave it to the Editor's discretion to make the acceptance decision.

 

Author Response

Dear reviewer,

First of all, thank you for the time you took to review the manuscript, we are sure that it will improve the standard of the work and we take good note of some suggestions for future research.

We agree that it is worthwhile to change the focus and talk about “maturity evolution” rather than “ovarian development”, to avoid false expectations about the content of the paper (L18, L85, L201), nevertheless it seems to us that the paper, with its applied approach, provides valuable results both for applied science and for the potential industry of this species, and provides knowledge at various levels about the reproductive biology of the species in captivity.

Regards.

Reviewer 2 Report

Comments and Suggestions for Authors

 The authors have thoroughly revised the manuscript and I see that revised manuscript looks promising. 

Author Response

Dear reviewer,

Thank you very much for taking the time to review the manuscript, which I am sure will enhance the quality of the manuscript.

Regards.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

I appreciate your intention to maintain a strong focus on your study about a restricted topic, it is your choice, for sure.

Despite this, I think that some comments might be more considered during your revision to enhance the soundness of your manuscript, but as stated it depends on your ideas.

 

Best regards

Author Response

Dear reviewer,

Our work is focused on aquaculture research and the work we develop in our group is directed to the development of zootechnical aspects with different species of commercial interest or with species with potential for the development of an industry in the medium term according to more sustainable characteristics (as is the case of C. labrosus). For this reason, more taxonomic or ecological aspects are beyond the scope of our study...

Nevertheless, we understand the interesting interaction between aquaculture research and other disciplines, and appreciate the time taken to review the manuscript and help improve the standard of the paper.

Regards.