Evaluation of Propylene Glycol and Essential Oil Supplementation on Growth Performance, Feed Efficiency, Serum Biochemical Indices, Hematological Parameters, and the Expression of Antifreeze IV and Lipid Metabolism-Related Genes in Nile Tilapia

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. For the following manuscript “Evaluation of propylene glycol and essential oils supplementation on growth performance, feed efficiency, serum biochemical indices, haematological parameters, and the expression of antifreeze-IV and lipid metabolism-related genes in Nile tilapia”

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

·        The introduction provides sufficient background

Are all the cited references relevant to the research?

Yes/Can be improved/Must be improved/Not applicable

·        The cited references relevant to the research

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

·        We clarify the research design appropriate based on the reviewer comment

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

·        The methods adequately described

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

·        We improved the results based on the reviewer’s comments

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

·        The conclusions supported by the results

3. Point-by-point response to Comments and Suggestions for Authors

Reviewer 1

1.Comments 1: [Table 3 (Page 260) could be removed, as water quality parameters should be included in the "Materials and Methods" section (Pages 148–152) rather than presented as a separate table]

1.Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have transferred the table in the materials and methods section under caption table 2. Also, the values of water quality parameters inserted in materials and methods section under the subtitles” Physicochemical parameters of water” – page 4, paragraph 5, and lines 153 to 158 the table in lines 219 to 211]

2.Comments 2: [On Page 144, it is stated that "Fish were fed experimental diets at a daily feeding rate of 5% of their live body weight for 6 days a week." Please clarify the rationale for feeding 6 days a week instead of 7 days, as this deviates from common aquaculture experimental protocols.]

Response 2: Thank you for pointing this out, and we apologize for the lack of clarity in the original text. [The six-day/ week feeding regime is a standard husbandry practice in our research facility, as it allows the cultured fish to consume any residual feed from previous days. Also, it helps maintain good water quality by minimizing waste accumulation in the system.

We added the following phrase in the manuscript “allowing fish to consume any residual feed ” page 4, paragraph 3, and line 143]

3.Comments 3: [The format of all tables should be standardized to the three-line table style, which is the preferred format for academic manuscripts.]

3.Response 3: Thank you for pointing this out. The format of all tables is changed to the three-line table style

4.Comments 4: [The explanation of error markers in the tables is insufficient. Specifically, it should be explicitly stated that direct comparisons cannot be made between the PG group and the EO group, to avoid misinterpretation by readers]

4.Response 4: [We thank the reviewer for this critical insight. The authors agreed that the use of superscripts was inappropriate and confusing.]

[The authors wish to clarify that our experimental design requires comparison across all five groups, including between the two substances (PG and EO), to fully address our research objectives. Therefore, we have maintained the superscript letters from one -way ANOVA with Post-hoc testing to show all valid treatment comparisons. Secondly, we added 3 columns to the right showing p-values from two-way ANOVA for the factors substance and concentration and interaction. Also, we added clarification in the footnote of the tables to make it more understandable.

can be found – page 8, and lines 260 to 262.]

5.Comments 5: [The error markers in Figures 1 and 2 are inappropriate, as these figures appear to imply comparability between the PG group and the EO group—this issue should be corrected, and corresponding clarifications added.]

5.Response 5: Thank you for pointing this out. We disagree with this comment. The error markers in figures 1 and 2 are the same in tables 4-8. With use of one-way ANOVA and multiple comparison among all groups, including comparison with the control and comparison among all treatments including the PG groups and the EOs groups to fully address our research objectives.

6.Comments 6: This study focuses on two individual additives (PG and EO), but no discussion is provided regarding combined supplementation of the two. Please address whether relevant studies on their co-supplementation have been conducted, and if so, summarize the key findings to contextualize the current research.

page 18- L640-648.]

6.Response 6: Thank you for pointing this out. We agree with this comment. Therefore, we added a paragraph at the end of the discussion about the combined supplementation of the two feed additives- page 18- L652-660.]

7.Comments 7: The Discussion section would benefit from being divided into several subsections (e.g., based on key research questions or outcomes) to enhance logical flow and readability. Additionally, it is recommended to include a summary figure that synthesizes the core findings of this study, as this would facilitate readers’ comprehension of the overall results and their implications.]

7.Response 7: Thank you for pointing this out. We agree with this comment. [we divided the discussion into several subsections based on key research outcomes page number: pages 13-16.

 Also, we added a summary figure at the end of the manuscript (Page 24).

4. Response to Comments on the Quality of English Language

Point 1:

Response 1:  we improved the quality of English along the manuscript. The changes are marked with red color.  

5. Additional clarifications

No comment

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. For the following manuscript “Evaluation of propylene glycol and essential oils supplementation on growth performance, feed efficiency, serum biochemical indices, hematological parameters, and the expression of antifreeze-IV and lipid metabolism-related genes in Nile tilapia”

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/

·        The introduction provides sufficient background

Are all the cited references relevant to the research?

Yes/Can be improved/Must be improved/Not applicable

·        The cited references relevant to the research

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

·        The research design appropriate

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

·        We improved the methods based on the reviewer’s comments

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

·                    We improved the results based on the reviewer’s comments

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

·        The conclusions supported by the results

3. Point-by-point response to Comments and Suggestions for Authors

Reviewer 2

1.Comments 1: [. Abstract: Some abbreviations, including MCV, MCH, Hb, and IGMH, do not have full names.]

1.Response 1: ] Thank you for pointing this out. We agree with this comment. [Full names of MCV, MCH, Hb, and IGMH are added to the abstract at page 1, 2 Line 45-50)

2.Comments 2: [I strongly suggest writing clearly whether the monosex Nile tilapia are female or male?.]

Response 2: [the monosex Nile tilapia were all males]. Thank you for pointing this out. We agree with this comment. Therefore, we have [added “all males” phrase in the revised manuscript: page 4, paragraph 1, and line 124.]

3.Comments 3: [What do the last three columns of Table 3 refer to?]

3.Response 3: [The 3 columns of table 3 were the p-value of main effects (Additives and their concentrations, but after revision, we prefer to remove it as there were no significant differences in physicochemical parameters of water, also we removed the table to the materials and methods section.]  page number 6, and lines 218 to221]

4.Comments 4: What is K (condition factor) in Table 4? And the author should write clearly how this data was obtained in the materials and methods section.

4.Response 4: The authors thank the reviewer for this comment and apologize for this oversight. [Condition Factor (K) was calculated to evaluate the welfare of the experimental fish at the end of the experiment period. It was calculated using the standard equation: K= 100 (W/L³). The formula was added to the revised manuscript in the “Biometric Indices Assessment” subtitle.] [page 4, paragraph 6, and line 162.]

5.Comments 5: [In Table 5, since there is no difference in FI, the letters indicating the difference should be removed. Line 246-247, the authors wrote that the groups received both doses of PG significantly (P≤0.05) have higher PER, EG, EU and CE compared with the control group and both doses of EOs. But according to Table 5, there is no significant difference in PER or EG between PG and EOs, so this description is not very accurate and needs to be revised.]

5.Response 5: [We thank the reviewer for their particular attention to detail and for identifying these inaccuracies. We have corrected both points. Firstly, we have removed the superscript letters from the Feed Intake (FI) row. Secondly, in the results section, we have completely rewritten the paragraph to provide an accurate description of our findings.

page 7, paragraph 4, and line: 246 to 251.]

6.Comments 6: [The authors mentioned that both doses of EOs and the low dose of PG significantly (P≤0.05) decreased ALT and AST levels (Line 273-274) and PG at low concentration unexpectedly significant increased AST (Line 274-275). According to these two sentences, the description about AST level change induced by PG at low concentration is contradictory and needs to be revised.]

6. Response 6: [The authors sincerely thank the reviewer for identifying this considerable contradiction in our text. According to the data in Table 7, it is clear that the second statement is the right one. We have therefore deleted the incorrect sentence and rewritten the results of AST and ALT to present precise and accurate findings. page 8, paragraph 2, and line: 271 to 274.]

7.Comments 7: [The discussion is too lengthy. I suggest streamlining the discussion section to highlight the key points.]

7.Response 7: Thank you for pointing this out. We agree with this comment.

[we divided the discussion into several subsections based on key research outcomes]  page number: pages 13-16.

8.Comments 8: [There are spelling mistake (line 70, “from”) and punctuation error (line 73, no period at the end of the sentence) in the manuscript. The first title (line 57 and 58) and the second title (line 119-120, 2. Materials and Methods) appeared twice. The lowercase letters in the second column of Table 8 should be superscripted. The gene names of SREBP1, FAS, and CD36 (line 342) should be italicized. In a word, the manuscript requires significant revisions for language and clarity.

8.Response 8:

§  spelling mistake (line 70, “from: corrected page 17-L71

§  punctuation error (line 73, no period at the end of the sentence): corrected Line 74  

§  The first title (line 57 and 58) and the second title (line 119-120, 2. Materials and Methods “repeated lines are removed.

§  The lowercase letters in the second column of Table 8 should be superscripted: corrected to superscripted.  

§  The gene names of SREBP1, FAS, and CD36 (line 342) should be italicized: All gene names along the manuscript are revised and turned to italic.

2. Materials and Methods) appeared twice: Thank you for pointing this out. We agree with this comment.: we deleted the repeated words pages 3, L 119.

4. Response to Comments on the Quality of English Language

Point 1:

Response 1:   we improved the quality of English along the manuscript. The changes are marked with red color.  

5. Additional clarifications

No comment

1. Comments 1: Line 159, “use” should be “by using”.

1.Response 1: Thank you for pointing this out.  Corrected to “by using”.

2.Comments 2: Line 162, in W/L3, 3 should be written as superscripts.

Response 2:  W/L3, 3 corrected to be superscripts.

3.Comments 3: Line 236, O. niloticus in the title of 3.1 should not be deleted.

3.Response 3:  Line 236, O. niloticus  ….. deleted

4. comment 4. In Table 4-7, the annotations for the last three columns of P-values are still unclear. For example, either PG or EO has two concentrations, what is the P-value of Conc, PG or EO, or both of them? What does “their interaction” mean?  

4.Response 4:     

·       P- value (Add.) means: the main effect of the Additive (PG vs. EO), regardless of the concentration.

·        P-value (Conc.) means: the main effect of Concentration (Low vs. High), regardless of the additives.

·        P-value (Add.*Conc.) means: the interaction between Substance and Concentration. Tests if the effect of concentration depends on the substance.

·        More clarification added to the heading  and the footnote of the three last columns in Tables 4-8, improved the reading of the tables and the corresponding results.