Effect of Siberian Ginseng Water Extract as a Dietary Additive on Growth Performance, Blood Biochemical Indexes, Lipid Metabolism, and Expression of PPARs Pathway-Related Genes in Genetically Improved Farmed Tilapia (Oreochromis niloticus)

Round 1

Reviewer 1 Report

There is no statistical analysi for the liver histology results. Description of liver histology relies only on subjective observations.

The broken line analysis (figure 1) is not described in material and methods. 

Author Response

Response to Reviewer 1 Comments

We thank the reviewer for all their insightful comments, which have helped us greatly improve the manuscript. Our responses to the reviewer’ comments are detailed below. We have reformatted the manuscript to meet Fishes style requirements, and refined the language of the manuscript.

Point 1: There is no statistical analysis for the liver histology results. Description of liver histology relies only on subjective observations.

Response 1: We used Image-Pro Plus (version 6.0.0.260, Media Cybernetics Corporation, USA) to measure the pixel area of lipid droplets (bright objects) in the image[1], and analyzed the differences in the pre area of lipid droplets between the control, 0.4‰ and 1.6‰ ASW groups by SPSS (Table 7) (lines 167-177, 262-263).

Table 7. Effect of ASW on hepatic fat deposition of GIFT (means ± SEM).

* Different superscript lowercase letters in the same row indicate significant difference (P < 0.05).

 Point 2: The broken line analysis (figure 1) is not described in material and methods.

Response 2: We have added a description of broken line analysis in “2.10. Data Analysis” (lines 202-203).

References:

1. Hu, Y.; Yu, J.; Cui, X.; Zhang, Z.; Li, Q.; Guo, W.; Zhao, C.; Chen, X.; Meng, M.; Li, Y.; et al. Combination Usage of AdipoCount and Image-Pro Plus/ImageJ Software for Quantification of Adipocyte Sizes. Front. Endocrinol. 2021, 12.

Author Response File: Author Response.docx

Reviewer 2 Report

First I would like to congratulate the authors on their manuscript. The manuscript is very well written, the introduction sufficiently presents the topic, and the results are described clearly and comprehensibly. I have only a few minor issues that I would like for the authors to address.

I recommend to changes some keywords, that are already included in the title.

References list should be improved, especially journal names. Some journals are written as a full name, the others are abbreviated. In the case of abbreviated ones, some are written with dots (for example no. 18) and the others without them.

Line 66 – please add a Latin name for zebrafish (Danio rerio)

Lines 113, 132 - change brackets from (35) to [35] and (36) to [36]. Also in line 132, I think it should be “Ma et al. [36]”. Similarly in line 145 – I think it should be “Qiang et al. [37]” instead of “Qiang, Wasipe (37)”. The same issues are in lines: 163, 204, 283

In Table 1 there is “Vitamin premix ^a” and “Mineral premix ^b”. What “a” and “b” superscripts mean?

Lines 117-119 - Fish were acclimated in an aerated flow-through system but during the supplementation period the water flow was changed to a circulating water system. Can you explain why water circulation was changed? It is a potentially stressful event for fish as it can change water parameters. The water parameters for the experimental period are in the manuscript, but what were the parameters in the acclimation period?

Line 157, 166 – add space in-between “5000g” and “4mm”

Line 169 – RT usually stands for Reverse Transcription and not Real Time. So consider changing “quantitative real-time PCR (qRT-PCR)” to “quantitative reverse transcription PCR (qRT-PCR)”

Line 92, 169 – in line 92 there is “PPARα, PPARγ” but in line 169 and later on these genes are written down differently (ppar α, ppar γ,). Please verify which version is correct.

Line 211 – the same information under Table 3, 5, 6 is written without bold text

Line 230 – “0.1‰, 0.2‰and 1.6‰” – lack of space between 0.2‰and

Line 272 – duplication -  0.74‰‰

Line 275 – I suggest removing one “and” form the sentence “and therefore are commonly used to evaluate fish nutrition and health and the stress response to the environment”

Line 297 – “[…] hypoglycemic effect as well [56, 57], and in our study, and in our study […]”

Lines 269 and 341 – in both sentences there is “yellow catfish” but with different Latin names. Please make sure that Latin names are correct.

Lines 382 – 395 - please standardise the way you write a colon - no space before, no bold

 Author Response

Response to Reviewer 2 Comments

We thank the reviewer for all their insightful comments, which have helped us greatly improve the manuscript. Our responses to the reviewer’ comments are detailed below. We have reformatted the manuscript to meet Fishes style requirements, and refined the language of the manuscript.

Point 1: I recommend to changes some keywords, that are already included in the title.

Response 1: Because Acanthopanax senticosus is also called Siberian ginseng, we have revised the title to avoid repetition of keywords (line 1).

Point 2: Line 66 – please add a Latin name for zebrafish (Danio rerio).

Response 2: We added the Latin name as suggested (line 62).

 Point 3: Lines 113, 132 - change brackets from (35) to [35] and (36) to [36]. Also in line 132, I think it should be “Ma et al. [36]”. Similarly in line 145 – I think it should be “Qiang et al. [37]” instead of “Qiang, Wasipe (37)”. The same issues are in lines: 163, 204, 283.

Response3: We changed brackets as suggested (lines 109, 136, 149, 168, 203, 299).

Point 4: In Table 1 there is “Vitamin premix ^a” and “Mineral premix ^b”. What “a” and “b” superscripts mean?

Response 4: We deleted the redundant superscripts "a" and "b" (Table 1).

Point 5: Lines 117-119 - Fish were acclimated in an aerated flow-through system but during the supplementation period the water flow was changed to a circulating water system. Can you explain why water circulation was changed? It is a potentially stressful event for fish as it can change water parameters. The water parameters for the experimental period are in the manuscript, but what were the parameters in the acclimation period?

Response 5: I am very sorry for the confusion caused by the difference in the meaning of words. In this experiment, the acclimation period and the experimental period of GIFT were cultured in the same system, in other words, the aerated circulation system and the circulating water system are the same system.

During the two (acclimation and experimental) periods, the water temperature was maintained at 28 ± 0.5°C, the pH was 7.4, and dissolved oxygen was ≥ 5 mg/L during two periods.

 Point 6: Line 157, 166 – add space in-between “5000g” and “4mm”

Response 6: We added space as suggested (lines 161, 169).

Point 7: Line 169 – RT usually stands for Reverse Transcription and not Real Time. So consider changing “quantitative real-time PCR (qRT-PCR)” to “quantitative reverse transcription PCR (qRT-PCR)”.

Response 7: We changed “quantitative real-time PCR (qRT-PCR)” to “quantitative reverse transcription PCR (qRT-PCR)” as suggested (lines 94, 178, 180).

Point 8: Line 92, 169 – in line 92 there is “PPARα, PPARγ” but in line 169 and later on these genes are written down differently (ppar α, ppar γ,). Please verify which version is correct.

Response 8: We normalized the version of genes in the article, capitalized and italicized.

Point 9: Line 211 – the same information under Table 3, 5, 6 is written without bold text.

Response 9: We have bolded the table information as you suggested (lines 213, 238, 251).

Point 10: Line 230 – “0.1‰, 0.2‰and 1.6‰” – lack of space between 0.2‰and.

Response 10: We added space as suggested (line 244).

Point 11: Line 272 – duplication - 0.74‰‰.

Response 11: We deleted the duplicate ‰ (line 288).

Point 12: Line 275 – I suggest removing one “and” form the sentence “and therefore are commonly used to evaluate fish nutrition and health and the stress response to the environment”.

Response 12: We changed this section to "and therefore are commonly used to evaluate fish nutrition, health, and the stress response to the environment" as suggested (line 292).

 Point 13: Line 297 – “[…] hypoglycemic effect as well [56, 57], and in our study, and in our study […]”

Response 13: We deleted the repeated sentence (lines 314).

Point 14: Lines 269 and 341 – in both sentences there is “yellow catfish” but with different Latin names. Please make sure that Latin names are correct.

Response 14: The Latin name of yellow catfish “Pelteobagrus fulvidraco” is correct, we have changed it (line 285).

Point 15: Lines 382 – 395 - please standardise the way you write a colon - no space before, no bold.

Response 15: We standardized the writing of colons as required (lines 397-410).

Author Response File: Author Response.docx

Reviewer 3 Report

The study by Li et al evaluated graded inclusion levels of water extracted Acanthopanax senticosus in the diets for Nile tilapia. The manuscript needs to be revised for English, and I have a few concerns regarding the statistical analysis and the feed formulation. Nile tilapia during the growthout phase can be fed 28% CP, but juveniles are fed diets with higher protein content (from 32% - 45%), which is questionable if these results can be translated to practical conditions.

Please find below my questions and suggestions:

L15: Efficient

L17: Promote lipid metabolism?

L20: Avoid inclusive words.

L21: What type of regression analysis? Linear? Quadratic?

L22: TG and TC are abbreviations not previously described.

L23: This sentence has to be improved. What do you mean by increasing and lowering HDL-C?

L25-29: In which organ were these genes upregulated?

L90: Avoid inclusive words.

L94: Avoid inclusive words.

L96: Avoid inclusive words. Please rephrase sentences with “we”.

L108-113: What is the reasoning for formulating diets for tilapia juveniles with 28% crude protein? This is definitely not what has been practiced by the industry. And what are the procedures to analyze the proximate composition of the diets?

Table 1: Are the feed formulas expressed as is or on a dry-matter basis? Please specify. Also, specify on the head of the table what the inclusion levels are. And lastly, where are the dry matter values for the diets?

L132: I imagine that one part had to have anti-coagulants. If so, which one was used and what concentration?

L163-166: What about the staining procedure?

L184-189: Why the procedures for the broken-line regression are not described here? And what were the criteria used to determine if the data was analyzed for comparison of means or regression?

Table 2: Abbreviations should be described in the footnote of the table.

Figure 1: R squared should be presented for the regression and specify if bars are standard deviation or standard error.

L240-241: This should be in M&M section.

L276-278: What do you mean by “granulocytes, monocytes, and lymphocytes that are closely related to the fish immunity”? 

Author Response

Response to Reviewer 3 Comments

We thank the reviewer for all their insightful comments, which have helped us greatly improve the manuscript. Our responses to the reviewer’ comments are detailed below. We have reformatted the manuscript to meet Fishes style requirements, and refined the language of the manuscript.

 Point 1: L15: Efficient.

Response 1: We removed the word (line 14).

Point 2: L17: Promote lipid metabolism?

Response 2: We modified this section to "regulate lipid metabolism" (line 15)

Point 3: L20: Avoid inclusive words.

Response 3: We changed the language as suggested (line 18).

Point 4: L21: What type of regression analysis? Linear? Quadratic?

Response 4: We used a "linear regression analysis" (line 18).

 Point 5: L22: TG and TC are abbreviations not previously described.

Response 5: We have supplemented the full names of TG, TC, HDL-C, LDL-C, SOD, and CAT in the “Abstract” (lines 20-23).

 Point 6: L23: This sentence has to be improved. What do you mean by increasing and lowering HDL-C?

Response 6: Sorry for the mistake here, the correct statement is "by increasing the high-density lipoprotein cholesterol concentration and lowering the low-density lipoprotein cholesterol concentration " (lines 21-22).

Point 7: L25-29: In which organ were these genes upregulated?

Response 7: Thanks for your suggestion, the organ is liver tissue, and we have added relevant information here (line 25).

Point 8: L90: Avoid inclusive words.

Response 8: We changed the language as suggested (line 85).

Point 9: L94: Avoid inclusive words.

Response 9: We changed the language as suggested (line 90).

 Point 10: L96: Avoid inclusive words. Please rephrase sentences with “we”.

Response 10: We changed the language as suggested (line 92).

 Point 11: L108-113: What is the reasoning for formulating diets for tilapia juveniles with 28% crude protein? This is definitely not what has been practiced by the industry. And what are the procedures to analyze the proximate composition of the diets?

Response 11: Thank you very much for your suggestion. It is true that adding 30%-40% crude protein to tilapia feed has the best growth performance. However, in recent years, the scale of tilapia aquaculture has expanded and the output has increased in China[1], and the price of tilapia in the market is less than 1 USD/ 500g. And the price of fish meal, the main source of protein in feed, continues to rise, leading to an increase in the feed cost as a proportion of the industry spending. Therefore, on the basis of not affecting the growth of tilapia, appropriately reducing the protein content is beneficial to the development of the industry.

Moreover, we also found that there are many studies [2–5] using feeds with similar crude protein content (28%-29%) for tilapia farming experiments, so this study still has certain study value for the development of tilapia feeds in industry. Thanks again for your suggestion, I will pay more attention to this aspect in future research.

We have supplemented the article with related methods for feed composition analysis (lines 109-116).

 Point 12: Table 1: Are the feed formulas expressed as is or on a dry-matter basis? Please specify. Also, specify on the head of the table what the inclusion levels are. And lastly, where are the dry matter values for the diets?

Response 12: The ingredients in the feed table have been air-dried before use, and we have added information on Dry matter in the table as you suggested (Table 1).

Point 13: L132: I imagine that one part had to have anti-coagulants. If so, which one was used and what concentration?

Response 13: In our study, no anti-coagulant was used. The blood was collected from the tail vein of GIFT and divided into two parts. One part was 20 μL added to 180 μL of dilution (including NaCl, Na₂SO₄, buffer, antibacterial solution, and so on), and analyzed in an automatic blood cell analyzer (bc-5300, MINDRAY, Shenzhen, China). The dilution and related kits were all purchased from MINDRAY.

 Point 14: L163-166: What about the staining procedure?

Response 14: We supplemented the detailed production process of liver section (lines 167-177).

 Point 15: L184-189: Why the procedures for the broken-line regression are not described here? And what were the criteria used to determine if the data was analyzed for comparison of means or regression?

Response 15: We added relevant content in “2.10. Data analysis”, the relationship between SGR and dietary ASW levels was determined using a two-slope broken-line model according to the method of Qiang, Khamis[4].

Point 16: Table 2: Abbreviations should be described in the footnote of the table.

Response 16: We have added a note in the footnote of the Table 2.

Point 17: Figure 1: R squared should be presented for the regression and specify if bars are standard deviation or standard error.

Response 17: We supplemented the R² of the trend line in Figure 1. And the relevant data of specific growth rate were provided in Table 1, which were expressed as (mean + SEM), so the bars in the figure represents the standard error.

Point 18: L240-241: This should be in M&M section.

Response 18: We removed that section (line 254).

Point 19:  L276-278: What do you mean by “granulocytes, monocytes, and lymphocytes that are closely related to the fish immunity”? 

Response 19: In our study, we found that 0.4‰ and 0.8‰ ASW could significantly increase the number of WBC in the blood of GIFT, and the WBC included granulocytes, monocytes, and lymphocytes, which are closely related to fish immunity, so we speculate that 0.4‰ and 0.8‰ASW may improve immune function of GIFT.

Reference：

1. China Fishery Statistical Yearbook China Fishery Statistical Yearbook; Beijing: China Agriculture Press, 2021;
2. Dawood, M.A.O.; Amer, A.A.; Elbialy, Z.I.; Gouda, A.H. Effects of Including Triticale on Growth Performance, Digestive Enzyme Activity, and Growth-Related Genes of Nile Tilapia (Oreochromis Niloticus). Aquaculture. 2020, 528, 735568, doi:10.1016/j.aquaculture.2020.735568.
3. Qiang, J.; Khamis, O.A.M.; Jiang, H.J.; Cao, Z.M.; He, J.; Tao, Y.F.; Xu, P.; Bao, J.W. Effects of Dietary Supplementation with Apple Peel Powder on the Growth, Blood and Liver Parameters, and Transcriptome of Genetically Improved Farmed Tilapia (GIFT, Oreochromis Niloticus). PLOS ONE. 2019, 14, e0224995, doi:10.1371/journal.pone.0224995.
4. Zeng, X.-N.; Zhang, X.-Y.; Chen, B.; Gao, L.-L.; Liu, S.; Wu, F.; Pan, Q. Dietary Four Different Woody Forages Differentially Affect the Growth, Feed Utilization, Apparent Digestibility, Intestinal Morphology and Microbiota Composition in Nile Tilapia (Oreochromis Niloticus). Aquac. Rep. 2021, 21, 100786, doi:10.1016/j.aqrep.2021.100786.
5. Qiang, J.; Yang, H.; Ma, X.Y.; He, J.; Wang, H.; Kpundeh, M.D.; Xu, P. Comparative Studies on Endocrine Status and Gene Expression of Hepatic Carbohydrate Metabolic Enzymes in Juvenile GIFT Tilapia (Oreochromis Niloticus) Fed High-Carbohydrate Diets. Aquac. Res. 2016, 47, 758–768, doi:10.1111/are.12534.

Author Response File: Author Response.docx