A Novel Approach to Perivitelline Fluid Extraction from Live Water-Activated Eggs from Zebrafish, Danio rerio

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript provides a detailed description of a method for collecting perivitelline fluid (PVF) from water-activated zebrafish eggs. According to the literature, including at least one of the cited articles, fixation in paraformaldehyde is generally required—particularly for unfertilized eggs and at the earliest developmental stages—in order to reduce the risk of rupture of the yolk or chorion and to minimize contamination. From this perspective, the method described here—allowing PVF collection from freshly activated eggs without fixation—could prove highly useful for future studies on PVF composition, which is increasingly recognized as important for embryonic protection and development. 

One potential concern, however, is the use of Milli-Q water as the activation medium. Due to its extreme purity and complete lack of ions, Milli-Q water is not physiologically representative of the natural conditions under which egg activation normally occurs. 

To the best of my knowledge, the precise mechanism of zebrafish egg activation is not fully understood. One possibility is that activation results from the dilution of serine protease inhibitors known to be expressed in fish ovaries. However, releasing the egg into a purely ionic-free medium might alter pH or other physicochemical conditions in a way that could affect activation dynamics. 

Was Milli-Q water chosen because it induces greater chorion swelling than other activation media? 

The authors provide convincing histological evidence supporting successful activation and PVF extraction. Nevertheless, I would be interested to know whether this method also works in a more physiologically relevant medium, allowing for downstream experiments involving PVF manipulation followed by fertilization and normal embryonic development. Milli-Q water, in this respect, seems suboptimal. 

Regarding Figure 5, at time point T8, where are the yolk proteins that are clearly visible at T0? T0 and T8 protein patterns appear surprisingly dissimilar. 

Finally, I suggest that immunoblots using selected antibodies would be more informative than Coomassie staining alone. For example, detecting 1–2 proteins known to be abundant in the yolk and in the chorion could help provide a clearer assessment of sample contamination. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Blake Lewis and colleagues reported a procedure for extracting perivitelline fluid from live, water-activated eggs of zebrafish (Danio rerio) using a micro-aspiration technique.

This manuscript is interesting. However, to be accepted in this journal, please address all the questions raised, provide more detailed information, include any additional data as needed, and revise the relevant figures.

This reviewer did not check for English language errors throughout the entire document; however, there are several typos. Please consult a professional editor to proofread the entire manuscript, including the figures and figure legends, before resubmission.

Comment:

1. In Figure 5, this reviewer believes that the proteins in the PVF originate from inside the eggs, such as from cortical granules. Therefore, they should also be present in the T0 sample. Did the authors detect the same proteins found in the PVF sample within the T0 sample? If so, why do the band patterns differ so markedly between the two? Why were the protein bands in the PVF sample not clearly detected in the T0 sample? Regarding the 15 kDa band observed in the PVF, what is its origin among the proteins present in the T0 sample? When increasing the amount of PVF protein loaded for SDS-PAGE analysis, do the band patterns become more similar to those of the T0 sample? 
2. Have you ever collected PVF from the eggs over time after activation and analyzed it by SDS-PAGE? Are the SDS-PAGE patterns of PVF proteins consistent across different time points?　
3. Have the authors ever detected any differences in the contents of the perivitelline fluid between samples collected from water-activated eggs and those collected from fertilized eggs following authors’ technology?

Please include the interpretation or relevant data in the Results or Discussion section. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The present methodological proposal, focused on the extraction of perivitelline fluid (PVF) from Danio rerio embryos activated by water, without the use of chemical fixatives, emerges as an attempt to improve experimental protocols aimed at the analysis of embryonic proteins. Instead of relying on the traditional formalin fixation—known for causing undesirable cross-linking in proteins—the authors seek to present an alternative that better preserves the integrity of the samples for proteomics applications. At first glance, this seems to be a promising approach. However, a closer reading reveals that the work presents significant gaps that limit its acceptance as a robust scientific contribution to fields such as developmental biology, reproductive toxicology, and functional proteomics.

From a thematic perspective, the article falls within a recognized field of relevance. The zebrafish (Danio rerio) has been widely used as a model in biomolecular studies due to its favorable characteristics, such as transparent embryos, high fecundity, and genetic proximity to higher vertebrates. PVF, in turn, is a biological matrix of increasing interest, not only because it contains maternal proteins crucial for embryonic survival, but also because it acts as a sensitive interface between the embryo and its environment. Despite this, the article fails to adequately explore the biological and translational potential of PVF. The description of its composition and function is superficial and does not address critical issues such as its role in maternal immunity transfer, regulation of embryonic developmental signaling pathways, or its function as a biomarker in ecotoxicological studies.

This omission undermines the conceptual framework of the proposal. There is a lack of connections to contemporary themes such as transgenerational epigenetics, systems biology, and biomarker-based proteomic platforms. Without this background, the work remains restricted to a technical perspective, failing to demonstrate a deeper understanding of the biological implications and real-world applications of the matrix under study.

Another critical point concerns the theoretical foundation. The authors mention the adverse effects of formaldehyde on protein structure but do not present quantitative data or cite relevant studies that concretely demonstrate such impacts. Moreover, the existence of alternatives already described in the literature—such as the use of less aggressive fixatives, stabilizing buffers, or even cryopreservation techniques—are ignored, which could achieve the same goal of the proposal with comparative advantages. What is missing here is a thorough, updated, and critical literature review that ties together the problem, scientific gap, and rationale of the proposed approach.

On the methodological front, while the technical description of the extraction procedure is clear, the experimental design is insufficient. There is no adequate biological replication, no control groups (such as the use of formalin for direct comparison), and no attempt at cross-validation with established techniques. The lack of reproducibility tests and the absence of any statistical analysis—even the most basic—render the presented data merely illustrative. The use of SDS-PAGE as an analytical tool is limited to visualizing bands, without quantitative analyses such as densitometry, loading control, or statistical comparisons between profiles.

The presentation of results is also lacking. The images lack informative captions, standardized scales, and critical interpretation. There is no discussion of intra- or interindividual variability, nor data on protein yield or embryonic integrity post-collection. A clear disconnection between the presented data and the broader scientific objectives of the study is evident. The figures, instead of complementing a consistent analysis, end up replacing analyses that would be expected in a work with more methodological rigor.

The discussion section, in turn, does not delve into the implications of the findings or confront them with specialized literature. The limitations of the protocol, such as the risk of contamination by yolk fluid, operational variability, or challenges in large-scale standardization, are not discussed. There is also a lack of perspectives for future research, such as mass spectrometry tests, using the protocol in other species, or long-term studies with proteomic profiling follow-up. The proposal, therefore, appears premature as a mature scientific contribution.

The conclusion does not contribute to reversing this scenario. Instead of providing a critical synthesis of the findings, it merely reaffirms the supposed advantages of the proposed technique without discussing its practical, ethical, or methodological limitations. The lack of self-reflection or mention of the need for independent validation weakens the potential applicability of the proposal in various laboratory contexts. Moreover, the work completely neglects the bioethical debate surrounding the manipulation of live embryos, which is particularly relevant in modern biomedical and toxicological research.

Finally, the article’s writing in English contains linguistic flaws that compromise its clarity and technical precision. There are fluctuations in the use of central terminology and an excess of passive constructions that dilute the experimental responsibility. These language problems indicate a lack of specialized revision and reduce the chances of acceptance in high-impact journals.

In summary, although the idea of a PVF extraction technique without the use of chemical fixatives represents a specific operational advancement, the article suffers from a lack of theoretical grounding, methodological weaknesses, and a lack of broader scientific contextualization. For the proposal to reach its potential as a real contribution to developmental biology and functional proteomics, a complete revision of the manuscript would be necessary, based on experimental rigor, connection to recent advances in the field, and attention to the ethical and analytical demands that guide contemporary science.

Comments on the Quality of English Language

A review of the article in English would be of great importance.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have responded convincingly to my comments and have improved the manuscript by clarifying several crucial aspects of the technique and the results presented. The introduction has also been enhanced, likely thanks to the input of the other reviewers. Overall, the manuscript is now suitable for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

Blake Lewis and colleagues reported on the procedure of perivitelline fluid extraction from live water-activated eggs from zebrafish, Danio rerio using the micro-aspiration technique. T This revised manuscript is both interesting and significantly improved. The authors have addressed the reviewers' comments to the best of their ability, making the manuscript much clearer. I recommend accepting it for publication.

 

Comments on the Quality of English Language

This reviewer did not check for English language errors throughout the entire document. Please consult a professional editor to proofread the entire manuscript, including the figures and figure legends, before resubmission.

Reviewer 3 Report

Comments and Suggestions for Authors

Boa noite

Venho por este meio informar que os autores atenderam às sugestões e correções solicitadas e indicadas, tornando o trabalho muito mais técnico, com linguagem científica muito melhor e referências atualizadas, tornando-o uma leitura mais interessante.