The Effect of Dietary Lipid Supplementation on the Serum Biochemistry, Antioxidant Responses, Initial Immunity, and mTOR Pathway of Juvenile Tilapia (Oreochromis niloticus)

Round 1

Reviewer 1 Report

Dear, Sir

          The manuscript is well-written; however, some minors are found. Could you please see the attached file?

Best regards,

Comments for author File: Comments.pdf

Well-done

Author Response

Response to Reviewer 1 Comments (Round 1)

Point 1: Fish possess natural antioxidant and nonspecific immune systems [5]. Unclear reason! and how?

Response 1: Thank you for the Reviewer's suggestion，the natural antioxidant and nonspecific immune systems of fish were antioxidant enzymes (superoxide dismutase, glutathione peroxidase, and catalase), nonspecific immune enzymes (lysozyme and alkaline phosphatase) and immune factors (tumor necrosis factor α, interleukin 1β, and interleukin 6). This sentence was mainly to lead to the following context, played a summary role.

Point 2: Researches? where are the propose of the researches?

Response 2: According to the Reviewer's suggestion, the “Researches” had been changed to “Previous studies had shown that”.

Point 3: Please add more investigations about these! and the authors should be warranted that your objective has never been proposed.

Response 3: Thank you for the Reviewer's suggestion，this part was a summary and investigation. Due to the limited number of references read by the authors, no similar views to the present paper have been found. In the future, the authors will strive to improve the research ability.

Once again, thank you very much for your comments and suggestion.

Reviewer 2 Report

review report attached

Comments for author File: Comments.pdf

a through revision nis essential.

Author Response

Response to Reviewer 2 Comments (Round 1)

Point 1: Abstract need to revise for better readability and to better understanding.

Response 1: According to the Reviewer's suggestion, the abstract had been revised.

Point 2: In the introduction, the authors could provide more information on the specific research gaps that this study addresses.  For example, what are the current knowledge gaps on the optimal dietary lipid levels for juvenile GIFT tilapia?  What are the mechanisms by which dietary lipid levels affect fish growth, immunity, and metabolism?

Response 2: According to the Reviewer's suggestion, this section had been added.

Point 3: The authors could also discuss the significance of their study in more detail.  For example, how can the findings of this study be used to improve the nutritional standards and commercial aquaculture production of juvenile GIFT tilapia?

Response 3: According to the Reviewer's suggestion, this section had been added.

Point 4: In the methods section, the author could provide more detail about how they collected and analyzed their data.  This would allow other researchers to replicate the study.

Response 4: According to the Reviewer's suggestion, this section had been added.

Point 5: The authors do not discuss the potential negative consequences of high lipid diets in fish.  For example, high lipid diets can lead to obesity, liver disease, and cardiovascular disease in fish.  The authors should discuss these potential negative consequences and acknowledge that more research is needed to determine the optimal lipid level for juvenile tilapia.

The authors do not discuss the implications of their findings for aquaculture practices.  For example, their findings suggest that increasing the lipid content in the feed could improve the growth and performance of juvenile tilapia.  However, the authors do not discuss the cost-effectiveness of this approach or the potential environmental impacts of aquaculture practices that use high lipid feeds.

Response 5: Thank you for the Reviewer's suggestion. Because the data in this paper did not involve growth performance, it was not described the potential negative consequences of high lipid diets in the discussion section. The growth performance of the animals has already been used in another paper, so the growth performance is not covered in this paper.

Point 6: The conclusion does not discuss the limitations of the study.  For example, the study did not measure the levels of specific fatty acids in the feed.  This is a significant limitation, as it limits the authors' ability to draw firm conclusions about the mechanisms by which lipids promote metabolic processes, protein absorption, and non-specific immune function in fish.

Response 6: According to the Reviewer's suggestion, the limitations of the study had been added.

Once again, thank you very much for your comments and suggestion.

Reviewer 3 Report

This study investigates the effect of different dietary lipid levels (0.35%-15.35%) on the serum biochemistry, antioxidant responses, initial immunity, and mTOR pathway of juvenile genetic improvement farmed tilapia. It shows that the dietary lipid content affects the serum biochemistry, enhances the antioxidant responses in the liver and serum, improves the nonspecific immune parameters in the serum, down-regulates the expression of inflammatory response genes, and up-regulates the expression of the mTOR. It is an interesting and well-conducted study, which could consider the following comments:

Major points:

The authors formulated their experimental diets with ingredients that were not common in practical diets, and that can affect the digestive process differentially. The authors would have to justify the chosen diet formulation since the ones used will not allow them to make direct inferences for aquaculture.

Although it is known that the optimal lipid content in the tilapia diet is between 7.7 and 9.3%, the authors consider the control diet to contain 0.35% lipids. The authors could justify this choice and they also could consider the effect of increasing lipids in the diet without having to consider a diet as the control.

Part of the content of the discussion (for example, lines 450 to 459, among other paragraphs) describes the function of the parameters studied, addressing introductory concepts that would be better placed in the introduction. The introduction has gaps that would be resolved.

The results could be rewritten, they are very repetitive in their structure. For example, lines 229-242 repeat practically the same structure with AST and ALT. If they were explained together, the text would be more agile and clearer. Something similar happens with lines 291 to 315 for TNF-α, Il-1β, and IL-6, and lines 336 to 356 for PIeK, AKT, and MTOR. In this sense, figures 1, 2, and 3 could be unified into 1A, 1B, and 1C with a single figure caption, and figures 4, 5, and 6 could be unified into 2A, 2B, and 2C. Figure 1 together with its figure foot could occupy one page and Figure 2 another. The argument would be easier to follow.

Both the key contribution and the conclusions should indicate the optimal amount of lipids that the tilapia diet should contain according to the study.

Incorporate the MDA reasonable levels into the discussion to avoid tissue damage.

The final weight of the animals in each condition could be indicated, this parameter could be discussed.

Minor points

At the bottom of Table 2, all acronyms should be included.

Line 121: What concentration of permanganate solution was used?

Line 132: the initial density of the fish could be indicated.

Line 144: Was MS-222 used at a concentration of 0.01 or 0.03%?

The results indicate that n is equal to 3, I understand that it refers to the number of tanks, but it could be indicated that each value comes from the average of 3 animals.

Lines 374 and 378: replace protein absorption with absorption of amino acids or peptides.

Lines 475-485 could be rewritten since two sentences partially repeat the same idea.

The title of quote 19 should be rewritten like the rest

Minor editing of the English language is required.

In lines 41-54, fish is repeated excessively.

Domesticated or domestication could be changed to acclimated or acclimatization.

Line 118: Culture could be changed to fish farming.

Author Response

Response to Reviewer 3 Comments (Round 1)

Point 1: The authors formulated their experimental diets with ingredients that were not common in practical diets, and that can affect the digestive process differentially.  The authors would have to justify the chosen diet formulation since the ones used will not allow them to make direct inferences for aquaculture.

Response 1: Thank you for the Reviewer's suggestion. The formulation we use is limited to experimental use because the ingredients are clear and it is easier to control experimental variables, for example, the amount of lipids added. The composition of commercial feed is not very clear, and it is impossible to control its composition. The feed formula with different dietary lipid levels were designed based on references and SC/T 1025-2004 tilapia feedstuff aquaculture industry standard (Ministry of Agriculture of the People’s Republic of China).

Point 2: Although it is known that the optimal lipid content in the tilapia diet is between 7.7 and 9.3%, the authors consider the control diet to contain 0.35% lipids. The authors could justify this choice and they also could consider the effect of increasing lipids in the diet without having to consider a diet as the control.

Response 2: Thank you for the Reviewer's suggestion. The choice of control group is also a limitation of this study. In the future, I will carefully consider its rationality in the formulation of experimental plans.

Point 3: Part of the content of the discussion (for example, lines 450 to 459, among other paragraphs) describes the function of the parameters studied, addressing introductory concepts that would be better placed in the introduction.  The introduction has gaps that would be resolved.

Response 3: According to the Reviewer's suggestion, part of the content of the discussion had been changed.

Point 4: The results could be rewritten, they are very repetitive in their structure.    For example, lines 229-242 repeat practically the same structure with AST and ALT. If they were explained together, the text would be more agile and clearer. Something similar happens with lines 291 to 315 for TNF-α, Il-1β, and IL-6, and lines 336 to 356 for PIeK, AKT, and MTOR. In this sense, figures 1, 2, and 3 could be unified into 1A, 1B, and 1C with a single figure caption, and figures 4, 5, and 6 could be unified into 2A, 2B, and 2C.    Figure 1 together with its figure foot could occupy one page and Figure 2 another.    The argument would be easier to follow.

Response 4: According to the Reviewer's suggestion, part of the content of the results had been changed.

Point 5: Both the key contribution and the conclusions should indicate the optimal amount of lipids that the tilapia diet should contain according to the study.

Response 5: According to the Reviewer's suggestion, the optimal amount of lipids had been added to the key contribution and the conclusions.

Point 6: The final weight of the animals in each condition could be indicated, this parameter could be discussed.

Response 6: Thank you for the Reviewer's suggestion. The final weight of the animals has already been used in another paper, so the growth performance is not covered in this paper.

Point 7: At the bottom of Table 2, all acronyms should be included.

Response 7: According to the Reviewer's suggestion, all acronyms had been included.

Point 8: Line 121: What concentration of permanganate solution was used?

Response 8: According to the Reviewer's suggestion, the juveniles were disinfected using a concentration of 1/100 000 potassium permanganate.

Point 9: Line 144: Was MS-222 used at a concentration of 0.01 or 0.03%?

Response 9: According to the Reviewer's suggestion, the concentration of MS-222 was 0.03%.

Point 10: The results indicate that n is equal to 3, I understand that it refers to the number of tanks, but it could be indicated that each value comes from the average of 3 animals.

Response 10: Thank you for the Reviewer's suggestion. N=3 means the number of tanks

Point 11: Lines 374 and 378: replace protein absorption with absorption of amino acids or peptides.

Response 11: According to the Reviewer's suggestion, the protein absorption had been changed to amino acids or peptides.

Point 12: Lines 475-485 could be rewritten since two sentences partially repeat the same idea.

Response 12: According to the Reviewer's suggestion, lines 475-485 has been rewritten.

Point 13: The title of quote 19 should be rewritten like the rest.

Response 13: According to the Reviewer's suggestion, The title of quote 19 had been rewritten.

Point 14: Domesticated or domestication could be changed to acclimated or acclimatization.

Response 14: According to the Reviewer's suggestion, the domestication had been changed to acclimatization.

Point 15: In lines 41-54, fish is repeated excessively.

Response 15: According to the Reviewer's suggestion, the fish had been changed.

Point 16: Line 118: Culture could be changed to fish farming.

Response 16: According to the Reviewer's suggestion, the culture had been changed to fish farming.

Once again, thank you very much for your comments and suggestion.

Round 2

Reviewer 2 Report

Now article can be accepted 

A through reading for spelling mistakes and gramatical mistakes is required 

Author Response

Response to Reviewer 2 Comments (Round 2)

Point 1: A through reading for spelling mistakes and gramatical mistakes is required.

Response 1: Thanks for the Reviewer's suggestion. According to the Reviewer's suggestion, the spelling mistakes and grammatical mistakes have been corrected. My native language is not English. Please point out the spelling mistakes and gramatical mistakes that have not been modified.

Best regards,

Once again, thank you very much for your comments and suggestion.

Reviewer 3 Report

Minor comments to the authors:

Line 257: n=3 could be replaced by n=3 tanks per condition.

Lines 430 to 440: “Lysozyme …infections [44]" should be removed, it has been moved to the introduction.

Line 720 to 728: These quotes have been moved forward, and should be deleted from here.

Line 63: had could be replaced by have.

Line 102: formula could be replaced by formulas.

Line 101: Was could be replaced by is.

Author Response

Response to Reviewer 3 Comments (Round 2)

Point 1: Line 257: n=3 could be replaced by n=3 tanks per condition.

Response 1: According to the Reviewer's suggestion, the n=3 of Table 1 to 7 and Figure 1 and 2 have been replaced by n=3 tanks per condition.

Point 2: Lines 430 to 440: “Lysozyme …infections [44]" should be removed, it has been moved to the introduction.

Response 2: According to the Reviewer's suggestion, the “Lysozyme …infections [44]" has been removed.

Point 3: Line 720 to 728: These quotes have been moved forward, and should be deleted from here.

Response 3: According to the Reviewer's suggestion, these quotes have been removed.

Point 4: Line 63: had could be replaced by have.

Response 4: According to the Reviewer's suggestion, the “had” has been changed to “have”.

Point 5: Line 63: had could be replaced by have.

Response 5: According to the Reviewer's suggestion, the “had” has been changed to “have”.

Point 6: Line 102: formula could be replaced by formulas.

Response 6: According to the Reviewer's suggestion, the “formula” has been changed to “formulas”.

Point 7: Line 101: was could be replaced by is.

Response 7: According to the Reviewer's suggestion, the “was” has been changed to “is”.

Once again, thank you very much for your comments and suggestion.