Effects of Heat-Induced Oxidative Stress and Astaxanthin on the NF-kB, NFE2L2 and PPARα Transcription Factors and Cytoprotective Capacity in the Thymus of Broilers

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigated the impact of heat stress (HS) on chicken development at the molecular level, focusing on the transcriptional changes induced in broiler thymuses. Additionally, it examined the potential mitigating effect of the antioxidant astaxanthin on HS-induced gene expression alterations. The findings indicate that HS upregulates the expression of genes involved in inflammation, oxidative stress responses, and apoptosis pathways. Supplementation with astaxanthin significantly reversed the transcriptional changes induced by HS in many of these genes. This suggests that astaxanthin might play a protective role against HS-induced damage through its influence on oxidative stress pathways.

 

While these transcriptional results provide valuable insights, it's crucial to note that the data are still very preliminary and do not yet strongly support the hypothesis.

 

Major comments:

 

1. A major concern regarding the growth outcomes is that the HSAX group did not exhibit a significant difference compared to the HS group, despite the authors noting a "slight" distinction. This finding does not strongly support a significant protective effect of astaxanthin against HS-induced developmental defects! Given that the transcriptional analyses focused on the thymus, it is essential to investigate whether HS and astaxanthin can induce phenotypical changes in the thymus, including changes in weight, size, physiology, pathology, cell morphology, and apoptosis. Such studies could clarify the potential protective role of astaxanthin in the thymus.

2. Please label the names of genes in Figures 3, 4, 5, and 6. These were not clarified in Results section either.

3. In Figure 4, the results indicate upregulation of all three genes in the Keap1-Nrf2 pathway. Since Keap1 is known as the primary repressor of Nrf2, increased Keap1 levels might induce degradation of Nrf2 protein despite enhanced gene expression. To further explore this, the study should investigate changes in Nrf2 protein levels using western blotting or immunostaining. Alternatively, examining a panel of classic Nrf2-target genes, including OH1, Gst genes, and Cytochrome genes, could validate the activation of the Nrf2 pathway.

4. A significant problem of the manuscript is the absence of in-depth discussions. The "Discussion" section merely summarizes the results without thorough explanation and exploration of potential working hypotheses and future directions based on the findings. The authors should expand on their interpretations and implications of the results to enrich the discussion section.

 

Minor comments:

1. Converting Table 4 into a figure with bar graphs is recommended to enhance the visibility of the results.

2. Table 5 presents information on differentially expressed genes sourced from databases, which duplicates information discussed in the "Discussion" section. Including these in a research publication may not add value.

 

Overall, this study presents only preliminary transcriptional analysis data without any mechanistic studies, detailed explanations, or deep discussions. It lacks substantial evidence supporting the "cytoprotective capacity" of astaxanthin in the chicken thymus.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study evaluated astaxanthin's protective effects against heat-induced oxidative stress and apoptosis in chicken thymus. And astaxanthin treatment mitigated these changes, preserving thymus health and function. The findings suggest astaxanthin's antioxidant potential in countering heat-induced inflammation and oxidative stress damage. However, there are some problems with the article.

1.      In this article, the number of individuals in each animal experimental group has not been specified, which is a crucial point and needs to be added.

2.      Additionally, there seems to be confusion regarding the relationship between the tables and figures, specifically between Table 3 and Figure 1. To streamline the presentation, it is recommended that only the results necessary for the main narrative be shown in the form of figures, as they are often more visually appealing and easier to comprehend. The tables, such as Table 3, can be included in an appendix or supplementary materials section to provide additional details without disrupting the flow of the article.

3.      The RT-PCR results should also be presented in a more visual format, such as bar graphs, rather than in tables. This will make the results more intuitive and easier to understand.

 

4.      In this paper, the four formats of Figure 3 and Figure 4 are relatively problematic and need further modification.

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have done their best to revise the manuscript. However, the revision mainly addresses the discussion section. None of the required experiments have been conducted, and the lack of samples cannot be an acceptable excuse. The absence of additional solid data cannot support the hypothesis and does not qualify this study for publication as a Research Article.

 

It is recommended to publish these preliminary results as a Brief Report/Communication instead of an article. The manuscript can be shortened by moving some materials, such as Tables 1, 2, and 5, to the Supplemental section.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study evaluated astaxanthin's protective effects against heat-induced oxidative stress and apoptosis in chicken thymus. And astaxanthin treatment mitigated these changes, preserving thymus health and function. The findings suggest astaxanthin's antioxidant potential in countering heat-induced inflammation and oxidative stress damage. However, there are some problems with the article.

1.      The RT-PCR results should also be presented in a more visual format, such as histogram, rather than in tables. This will make the results more intuitive and easier to understand. Presented as a figure instead of a table.

2.      In Figure 3B, there are no error bars, and the standard deviation observed in this study was relatively high, for example, Figure 3B, Figure 4A, and Figure 5b, indicating a need for further replication of the experiments.

 

3.      This version is not changed from the last version, please modify as required

Comments on the Quality of English Language

 Moderate editing of English language required

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

This study evaluated astaxanthin's protective effects against heat-induced oxidative stress and apoptosis in chicken thymus. And astaxanthin treatment mitigated these changes, preserving thymus health and function. The findings suggest astaxanthin's antioxidant potential in countering heat-induced inflammation and oxidative stress damage. However, there are some problems with the article.

 

The article has been revised to address the previous issues raised, but the arrangement of the images can still be further adjusted for better presentation.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Comments 1: The article has been revised to address the previous issues raised, but the arrangement of the images can still be further adjusted for better presentation.

Response 1: Point-for-Point Response: Arrangement of the images.

1. Thank you for the recommendation to consider the order of the images. Figure 2 is now the Gene Expression Heat Map, and Figure 3 is now the histogram of the Gene Expression Across Conditions. We believe this order will better provide for visual expression of the data Table 2 of the differentially expressed genes.