Effects of Fishmeal Replacement by Clostridium Autoethanogenum Protein Meal on Cholesterol Bile Acid Metabolism, Antioxidant Capacity, Hepatic and Intestinal Health of Pearl Gentian Grouper (Epinephelus Fuscoguttatus ♀ × Epinephelus Lanceolatus ♂)
Round 1
Reviewer 1 Report
General comments:
The integrity of the paper is good on the whole, but the major problem of the paper is that there are many mistakes in wordings and syntax and the rigor of the discussion is not good, which is required to be improved greatly.
Specific comments:
(1) There are many wordings and syntax of the text that cause unsmooth reading. For example, line 33: The sentence is incomplete and an unclear expression, i.e., a decrease of growth at what level of CAP.
(2) line 52-53: the phrase "the biological proteins processed by bacteria and yeast" inaccurately defines the single-cell protein; line 389-390: an incomplete expression; line 412-413: can't understand this sentence, etc. Please check and polish the paper to make it more readable.
(3) line 69-71: As a protein ingredient, insufficient phosphorus and lipid of CAP are regarded as a disadvantage of CAP, which is not proper cognition in itself.
(4) Material and methods section:
Please list the crude and fat levels of fish meal, CAP, soybean meal, gluten and wheat flour and describe the type of grain from which the protein material comes. Please list the CAP manufacturer.
Please provide the sequence numbers of genes used for real-time PCR (Table 2).
table 7 and figure 1: A clear distinction should be made between the observations of light microscopy and electron microscopy.
line 387-392: Please note that "fishmeal replacement or fishmeal was replaced" here is different from "fishmeal protein replacement or fishmeal protein is replaced". The expressions of fishmeal replacement or fishmeal protein replacement should be consistent across all the text.
(5) Discussion section:
line 431-432: Please note that the hepatic apoptosis was not investigated in this study.
line 438-452: an unconvincing analysis of associations of dietary CAP levels and lipid metabolism. The same is true for the effects of CAP on other indices. A comprehensive discussion is needed to have the rationality of fishmeal replacement by CAP in response to the growth performance of the fish species and its underlying metabolic mechanism at both biochemical and biomolecular levels.
(6) Inclusions: Please make comments on the application prospect of this new protein ingredient in aquatic feed.
Author Response
Response to Reviewer 1 Comments
Dear Reviewers，
We are very sorry, while checking we found that our response was not attached. Here are the results of the re-upload.
Point 1: There are many wordings and syntax of the text that cause unsmooth reading. For example, line 33: The sentence is incomplete and an unclear expression, i.e., a decrease of growth at what level of CAP.
Response 1: Thank you for your valuable comment, we have made the correction.(line 33-36) (The line number indicates the location in this attachment)
Point 2: line 52-53: the phrase "the biological proteins processed by bacteria and yeast" inaccurately defines the single-cell protein; line 389-390: an incomplete expression; line 412-413: can't understand this sentence, etc. Please check and polish the paper to make it more readable.
Response 2:
Line 52-53: the phrase "the biological proteins processed by bacteria and yeast" inaccurately defines the single-cell protein.
Thank you for your suggestion, we have corrected it to “the biological proteins processed by microalgae, fungi, and bacteria”.(line 55-56)
Line 389-390: an incomplete expression.
Thank you for your valuable comment, we have made the correction.(line 441-443)
Line 412-413: can't understand this sentence, etc.
Thank you for your valuable comment, we have made the correction.(line 464-468)
polish the paper to make it more readable
We first revised the paper by taking into account the comments of three reviewers. After that we checked and polish the paper.
Point 3: line 69-71: As a protein ingredient, insufficient phosphorus and lipid of CAP are regarded as a disadvantage of CAP, which is not proper cognition in itself.
Response 3: Thank you for your valuable comment, we have amended this sentence.(line 72-73)
Point 4: Material and methods section:
(1)Please list the crude and fat levels of fish meal, CAP, soybean meal, gluten and wheat flour and describe the type of grain from which the protein material comes. Please list the CAP manufacturer.
(2)Please provide the sequence numbers of genes used for real-time PCR (Table 2).
(3)table 7 and figure 1: A clear distinction should be made between the observations of light microscopy and electron microscopy.
(4)line 387-392: Please note that "fishmeal replacement or fishmeal was replaced" here is different from "fishmeal protein replacement or fishmeal protein is replaced". The expressions of fishmeal replacement or fishmeal protein replacement should be consistent across all the text.
Response 4:
(1) Thank you for your valuable comment, we have made additions.(line 95-102)
(2) Thank you for your valuable comment, we have made additions.(line 210-212, table 2)
(3) Thank you for your valuable comment, we have amended.(line 327-376, table7, table8, figure 1, figure2)
(4) Thank you for your valuable comment. After some deliberation, we determined that 15-60% of the fishmeal was replaced, so the correct expression should be "replace fishmeal". The corresponding part of the text has been replaced.
Point 5: Discussion section:
line 431-432: Please note that the hepatic apoptosis was not investigated in this study.
line 438-452: an unconvincing analysis of associations of dietary CAP levels and lipid metabolism. The same is true for the effects of CAP on other indices. A comprehensive discussion is needed to have the rationality of fishmeal replacement by CAP in response to the growth performance of the fish species and its underlying metabolic mechanism at both biochemical and biomolecular levels.
Response 5 :
(1) Thank you for your valuable comment, we have removed this inconclusive conclusion.(line 495-499)
(2) Thank you for your valuable comment, we have revised according to your suggestions. (line 500-525, line 554-555, line 544-545) This experiment is a preliminary exploration of the effects of CAP on grouper, the feeding trial is 8 weeks, so there maybe some differences in growth performance and physiological responses of grouper. We have revised the discussion and we believe that our findings will help us to conduct more in-depth studies in the future, such as designing more precise experiments to investigate the mechanism of cholesterol metabolism, or the repair of intestinal and liver health problems, or the mechanism of epinecidin production and the effect of bacterial challenge..
Point 6: Inclusions: Please make comments on the application prospect of this new protein ingredient in aquatic feed.
Response 6: Thank you for your valuable comment, we have made additions.(line 585-588)
Author Response File: 
Author Response.docx
Reviewer 2 Report
The present research investigated the feasibility and suitable substitution amount of fishmeal in the feed of pearl gentian grouper by replacing with Clostridium autoethanogenum protein meal, and has important theoretical and applied significance. However, the manuscript has various weaknesses and need to be improved.
1. There are language and technical mistakes in the presentation of the manuscript. Author should check and improve the manuscript.
2. Abstract should correctly reflect the main results of the research. The description about growth performance of fish in Abstract is incomplete (lines 33-34). Author made the analysis of gene expression and tissue morphology, but it had no relevant results in Abstract.
3. Define and explain all abbreviations on first appear in the text, make sure that the presentation is correct and consistent.
4. In Introduction section, the background of the effects of dietary CAP on fish is rarely introduced.
5. There is insufficient information given about the study design and analysis in M&Ms section, and some information are imprecise. In this experiment, total 20 tanks were used. But there were five experimental diets and each diet was assigned to three tanks (lines 112-113). Please check this procedure. What biochemical indicators are analyzed in each tissue should be described clearly. Which metabolic pathway gene expression is analyzed should be described clearly.
6. In results, the description of some results was inconsistent with the data in table and figure, especially in 3.4 and 3.6 sections. Therefore, it is hard to understand these results fully and correctly.
7. Please check the word of “shrimp” in line 352.
8. Please correctly write the Latin name of species. The Latin name of species should be written in full for the first time mention in manuscript, then the shortened Latin name should be written.
9. Why did author cite the reference of 23 to discuss the palatability of CAP? The reference studied the effects of yeast protein not CAP (line 390).
10. Author did not discuss the results well. Relevant parameters should be discussed together, such as the gene in Nrf2-Keap1 signaling pathway affects the oxidative stress, should be discussed in conjunction with antioxidant enzymes not ACP, etc. The authors need to discuss results clearly and logically.
Author Response
Response to Reviewer 2 Comments
Point 1: There are language and technical mistakes in the presentation of the manuscript. Author should check and improve the manuscript.
Response 1: Thank you for your suggestions. We have made the changes after carefully considering your suggestions and those of the two reviewers.
Point 2: Abstract should correctly reflect the main results of the research. The description about growth performance of fish in Abstract is incomplete (lines 33-34). Author made the analysis of gene expression and tissue morphology, but it had no relevant results in Abstract.
Response 2:
Thank you for your suggestion, we have corrected the problem.(lines 34-36)
Unfortunately, due to the large number of studies and the word limit of 200 words in the abstract, we had to abandon the presentation of the results for tissue morphology and some of the gene expressions.
Point 3: Define and explain all abbreviations on first appear in the text, make sure that the presentation is correct and consistent.
Response 3: Thank you for your suggestion, we have corrected the problem.(line 19, lines 40-42)
Point 4: In Introduction section, the background of the effects of dietary CAP on fish is rarely introduced.
Response 4: Thank you for your valuable comment, we have made additions.(lines 81-86)
Point 5: There is insufficient information given about the study design and analysis in M&Ms section, and some information are imprecise. In this experiment, total 20 tanks were used. But there were five experimental diets and each diet was assigned to three tanks (lines 112-113). Please check this procedure. What biochemical indicators are analyzed in each tissue should be described clearly. Which metabolic pathway gene expression is analyzed should be described clearly.
Response 5: Thank you for your valuable comment, we have made the correction.(line 127, lines 159-178, lines213-210)
Point 6: In results, the description of some results was inconsistent with the data in table and figure, especially in 3.4 and 3.6 sections. Therefore, it is hard to understand these results fully and correctly.
Response 6: Thank you for your valuable comment, we have made the correction.(lines 315-316, lines 381-382)
Point 7: Please check the word of “shrimp” in line 352.
Response 7: Thank you for your valuable comment, we have made the correction.(lines 403-404)
Point 8:Please correctly write the Latin name of species. The Latin name of species should be written in full for the first time mention in manuscript, then the shortened Latin name should be written.
Response 8: Thank you for your valuable comment, we have made the correction.(lines21-22, lines 438-441)
Point 9:Why did author cite the reference of 23 to discuss the palatability of CAP? The reference studied the effects of yeast protein not CAP (line 390).
Response 9: Thank you for your valuable comment. Initially, we thought that yeast protien and CAP are both products of alcoholic fermentation and both are single-cell protein that may have commonality. However, after further consideration, we decided to delete the relevant part because we thought it was not theoretically sound after all.(line 441-443)
Point 10: Author did not discuss the results well. Relevant parameters should be discussed together, such as the gene in Nrf2-Keap1 signaling pathway affects the oxidative stress, should be discussed in conjunction with antioxidant enzymes not ACP, etc. The authors need to discuss results clearly and logically.
Response 10: Thank you for your valuable comment, we have made a revision according to two logics. The first one is to try to discuss Relevant parameters together, and the second one is to try to discuss indicators of the same organization together. This was done as a combination to reduce the number of times similar summary statements occur.
Author Response File: 
Author Response.pdf
Reviewer 3 Report
The objective of the MS is to determine the suitability of Clostridium autoethanogenum meal as a fishmeal substitute in feeds for pearl gentian grouper. The subject is interesting and deserves to be studied, considering the current need to replace fishmeal in fish feeds.
The design and experimental procedures are adequate. A wide range of parameters related to growth, food utilization, metabolic and antioxidant status, gene expression, and tissue morphology are analyzed, allowing conclusive results.
I have some comments and minor corrections:
Throughout the manuscript: replace "intestines" with "distal intestine"
Pg 6, Ln 209: the name of the multiple comparison tests is Tukey, not Turkey
Pg 8: The units of expression of both MDA and GSH in liver and distal intestine is not correct. Referring those parameters to mg of protein makes no sense. They are usually expressed per gram of tissue.
Author Response
Response to Reviewer 3 Comments
Point 1: Throughout the manuscript: replace "intestines" with "distal intestine"
Response 1: Thank you for your valuable comment, We chose distal intestine section to ensure consistent sampling sites to reduce errors, however, in deference to the intent of the reference, we were not able to modify some of the word "intestine" in the text. We have made the following changes:Pg 17, Ln 569; Pg, 18, Ln 573 and Ln 580
Point 2: Pg 6, Ln 209: the name of the multiple comparison tests is Tukey, not Turkey
Response 2:
Line 52-53: Thank you for your suggestion. This was our mistake. We have made corrections.(Pg6, Ln 245)
Point 3: Pg 8: The units of expression of both MDA and GSH in liver and distal intestine is not correct. Referring those parameters to mg of protein makes no sense. They are usually expressed per gram of tissue.
Response 3: Thank you for your suggestion, please allow us to explain: we use (nmol/mgprot) or (μmol/mgprot) as the unit of MDA or GSH by referring to the kit instructions. Since the sampling technique cannot be the same every time, such as the amount of saline residue after washing the blood and mucus on the surface of the tissue, we cannot guarantee the same purity of the tissue samples, but this error can be eliminated to some extent by testing the protein content. In addition, unfortunately, we did not keep the original data of the tissue mass weighed during tissue homogenization, so we cannot convert the data to (nmol/g) or (μmol/g) now.
Here are some references, the MDA content is expressed in (nmol/mgprot):
https://doi.org/10.1111/are.14446
https://doi.org/10.1016/j.aqrep.2021.100938
https://doi.org/10.1016/j.aqrep.2021.100862
https://doi.org/10.3389/fmars.2021.667432
Here are some references, the GSH content is expressed in (μmol/mgprot):
https://doi.org/10.1016/j.fbio.2022.102330
https://doi.org/10.3390/jmse10101544
https://doi.org/10.1111/ejn.15735
https://doi.org/10.1016/j.actaastro.2020.02.007
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The manuscript is in better condition in this version, and can be accepted for publication.
Author Response
Thank you very much for your comments!
