Round 1

Reviewer 1 Report

The manuscript (ID: fishes-2401230) describes an experiment aimed at evaluating the effects of replacing fishmeal in gilthead seabream diets using three organic raw materials (the rest of rainbow trout, visceral Iberian pig and insect meal) on the growth performance,  nutritional parameters,  biometric indices, body composition, retention efficiency,  digestibility,  and histology of liver and intestine. The subject itself is surely worthy of investigation. However, some points need to be addressed as follows:

1)      L25-27: The conclusion section should be rewritten, as in the current form, it is only a general statement. The conclusion should answer the aim of the study.

2)      The introduction is written in short paragraphs, which you may collect into three to four paragraphs. Also, the hypothesis of the study should be clarified at the end of the Introduction section.

3)      Throughout the manuscript, the abbreviation must be introduced upon the first mention of the full term followed by its abbreviation in parentheses: From then on, the abbreviation must be used exclusively and throughout.

4)      L114: Add more descriptions about the experimental tanks, such as their type and dimension.

5)      L114: The average initial weight of fish (97 g) does not concur with the data in Table 4 (from 89 g to 96.5 g). Please add the overall mean ± standard error.

6)      L117-119: the ranges of all physical and chemical parameters throughout the trial period should be given.

7)      Throughout the M&Ms section, the instruments used must contain all full information (i.e., model, company, city, country).

8)      L119: What duration was the photoperiod during the experiment?

9)      L125-127: How were the organic raw materials used in this trial produced and what is their source?

10)  L132-133: Add a reference for Regulation (EU) 2018/848.

11)  The authors had to justify on what basis they selected the level of the tested organic raw materials (TRO, INS, and IBE).

12)  In all Tables: describe the experimental groups in the tables' footnotes. Also, add a column in all tables for the exact p-value.

13)  Table 1: add the calculated gross energy content of the experimental diets.

14)  In Table 1 footnote, two premix compositions were provided. which one is correct? Why weren't minerals included in the premix?

15)  L159: Why was the fish weighed in each tank individually? Will this cause stress for fish? Are the fish marked to follow them individually?

16)  L203: What percentage of chromium oxide was used in diets?

17)  Table 3: Replace “Ph” with “pH”.

18)  Table 4: Replace “ESM” with “SEM”.

19)  In Tables 5 and 6, The units are missed for all parameters.

Moderate editing of the English language.

Author Response

Response to Reviewer 1 Comments

Dear Editor

We have carefully read the reviewers’ comments regarding the manuscript recently submitted to Fishes," New organic raw materials for Gilthead seabream (Sparus aurata) feeding effects on growth, nutritive parameters, digestibility, and histology.” Below we respond to each of the suggestions by the reviewers. The manuscript has been thoroughly revised in response to the reviewers' suggestions. The revised manuscript has been provided to a native English-speaking translator for review. We will re-upload a manuscript that responds to comments and changes and hopes that the revised manuscript will be published in FISHES smoothly.

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Reviewer 1.

Point 1: L25-27: The conclusion section should be rewritten, as in the current form, it is only a general statement. The conclusion should answer the aim of the study.

Response 1: Thanks to the reviewers for their suggestions; we have rewritten the conclusion section; please refer to LINE 25.

Point 2: The introduction is written in short paragraphs, which you may collect into three to four paragraphs. Also, the hypothesis of the study should be clarified at the end of the Introduction section.

Response 2: Revised and corrected. For the hypothesis of the study, please refer to line 106.

Point 3: Throughout the manuscript, the abbreviation must be introduced upon the first mention of the full term followed by its abbreviation in parentheses: From then on, the abbreviation must be used exclusively and throughout.

Response 3: Thanks to the reviewers for their suggestions; we have revised all abbreviations throughout the manuscript.

Point 4: L114: Add more descriptions about the experimental tanks, such as their type and dimension.

Response 4: revised and added line 125. Concrete ponds with octagonal shapes.

Point 5:      L114: The average initial weight of fish (97 g) does not concur with the data in Table 4 (from 89 g to 96.5 g). Please add the overall mean ± standard error.

Response 5: Thanks to the reviewers for their suggestions; it was an error, and we have corrected it. Line 129. Overall mean ± standard error was added.

Point 6: L117-119: the ranges of all physical and chemical parameters throughout the trial period should be given.

Response 6: All physical and chemical parameters were added throughout the trial, lines 135/136.

Point 7: Throughout the M&Ms section, the instruments used must contain all full information (i.e., model, company, city, country).

Response 7: Throughout the M&Ms section, the instruments are revised and contain full information(i.e., model, company, city, country).

Point 8: L119: What duration was the photoperiod during the experiment?

Response 8: the photoperiod duration during the experiment (August to November 2021) was about (~ 12.5 h) and added line 137.

Point 9: L125-127: How were the organic raw materials used in this trial produced, and what is their source?

Response 9: this paragraph was added to answer your question lines 142-148. All the organic ingredients come from organic certified producers with the EU organic label. Trout was self-processed to extract the meat slices, and just the remaining parts were carefully cut, oven-dried, and ground into a suitable form for use in the feed. The remaining parts used for the Iberian pig meal were the liver, intestines, and heart, which were cut into small pieces. After oven drying, they were ground into a suitable form for incorporation into the feed. The insect component, larval insects, was utilized. They were received already dried and then ground for inclusion in the diets.

It should be noted that the sources of the insects and visceral Iberian pigs used in the study are kept confidential.

Point 10: L132-133: Add a reference for Regulation (EU) 2018/848.

Response 10: The Regulation (EU) 2018/848 reference was added to line 153.

Point 11: The authors had to justify on what basis they selected the level of the tested organic raw materials (TRO, INS, and IBE).

Response 11: Added in L 149 The level of inclusion of the TRO, IBE, and INS were fit to replace fishmeal and maintain total protein constant.

Point 12: In all Tables: describe the experimental groups in the tables' footnotes. Also, add a column in all tables for the exact p-value.

Response 12: The description of the experimental groups was added in the tables' footnotes, and a column for the exact p-value was added throughout the manuscript.

Point 13: Table 1: add the calculated gross energy content of the experimental diets.

Response 13: The calculated gross energy content of the experimental diets was added to Table 1.

Point 14:   In Table 1 footnote, two premix compositions were provided. which one is correct? Why weren't minerals included in the premix?

Response 14: We have used a multivitamin and minerals mix. he two premixes are correct we used a mix of them.

Point 15: L159: Why was the fish weighed in each tank individually? Will this cause stress for fish? Are the fish marked to follow them individually?

Response 15: Thanks to the reviewers for their comments; it was an error, I have revised the protocol and just the initial and final weighed was done individualle, the  others were weighed in groups of 5 fish not individually, to decrease the animal stress and corrected in the manuscript line181. In initial and final monitored the individual weight is useful for us because it give as information about dispersion, the intemedial wieghed are used to control mortality and biomass, so individual wieght is not useful for us.

Point 16: L203: What percentage of chromium oxide was used in diets?

Response 16: The percentage of chromium oxide was (50 g kg^-1) and added line 228.

Point 17: Table 3: Replace “Ph” with “pH”.

Response 17: Revised and corrected.

Point 18: Table 4: Replace “ESM” with “SEM”.

Response 18: Revised and corrected.

Point 19: In Tables 5 and 6, The units are missed for all parameters.

Response 19: Revised and added in the table title. The units were %.

Comments on the Quality of the English Language: Moderate editing of the English language.

A native English-speaking teacher has reviewed this manuscript to ensure the integrity of grammar and wording. I hope that the revised manuscript can be successfully published in FISHES.

To conclude, we thank the reviewers for their comments/suggestions, as they allowed us to improve the manuscript greatly.

Sincerely yours,

The authors

Author Response File: Author Response.pdf

Reviewer 2 Report

Interesting paper, excellent multidisciplinary approach. Innovative idea to recover waste from organic production to replace fishmeal and move this sector towards increasing environmental sustainability, also with a view to a circular economy.

Author Response

Thank you for reviewing the document. We appreciate your time and effort in providing feedback. However, we didn't receive any comments or suggestions.

Reviewer 3 Report

Dear Authors

The ms entitled "New organic raw materials for Gilthead seabream (Sparus aurata) feeding effects on growth nutritive parameters, digestibility, and histology." is an interesting, well-done, and carefully research paper that provides information along with the organic culture of gilthead seabream.  

The results presented in this study are interesting and well-structured and provide some good initial research results combining and managing to improve organic feed formulation in organic fish farming.

In addition to these general points, there are some specific points which are addressed as follows:
1. Throughout your manuscript (abstract, introduction, results, discussion, and conclusion), please where it is written seabream, and replace it with "gilthead seabream."

2. Line 54, Gambelli et al. [4], please rewrite as According to Gambelli et al. [4] …..and reword the whole sentence

3. Line 69-70: A few studies ……., please add a reference. 

Also, in the same Line 69:  Li et al. […] is missing a reference and should be written

or rewritten -rephrase the above sentence on the following lines, line 69 to line 72 (Li et al. [...],...... to growth performance)

4. Line 76 Another study by Khieokhajonkhet et al. [the reference is missing] ….,

It is suggested to rewrite it as "Previous studies evaluated the use of ….., and protein utilization [11]

5. Line 92  Another study… please rewrite as "On the other hand, it was reported by  Estevez et al. [3] that, to evaluate new organic ingredients, such as green pea protein and brown seaweed, to replace fish meals in feeds suitable for organic production.

6. Line 110-111 "For that purpose." Please rewrite it: " In total, were used 300 fish with an average mean weight of 97 g ± SEM and average mean length ……, and were distributed in 12 tanks of 4000L volume.

7. Line 113 “ after ten days adaptation,”  the adaptation period is not too short; please explain briefly.

8. Line 114-115 “The trial was within a saltwater recirculating system of ….” Please explain. Also, in the same lines you write of 75 m3, please add m3 as m³

In the same lines, the number of individuals that were added in the tanks is unclear. Please add it.

9. Line 117-119: “ Additionally,……, and pH.” It would be best to write the water quality ranges for the parameters that you mentioned in the ms (DO, ToC, ammonium, nitrate, nitrite, salinity, and pH) and whether they were kept constant throughout the experiment since you have not included water quality in the results.

10. Lines 123-127, The replacement was 100%? Please write it.

11. Line 124: please delete the space and write it as “ ^oC.”

12. Line 165: Briefly explain why was sampled three fish from each tank.

13. Line 194-197: “To measure the biometric parametric of the fish,” the fish were anesthetized. Please explain (the anesthetic that was used and the dose)

13. Line 199-201:Please give us more information in detail about the experiment. Was it a different experiment that was conducted in another RAS? Correct?

14. Lines 219 -224,  “Please explain briefly,” Were the fish being sacrificed?

15. Table 4 “ESM”  do you mean "SEM"? Please replace it. Also, in Table 4,"final weight" the letters "a, b" indicate significant differences to be rewritten in exponents.

16. In Tables 5, 6, 7 8,9, 10. the letters "a, b" indicate significant differences to be rewritten in exponents.

17. Line 358 “table 10”, maybe it's table 9". Please renumber the table and within the text

17. Line 377 Table 11”  Table 10??? Renumber the table and within the text.

18. Figure 2 and Figure 3 provide a better analysis of the graph

19. Lines 414, “In 2017, ….”, please rewrite it as” According to Di Marco […reference ] the organic……..

20. Lines 421 “Mende et al.”  Please rewrite it as Mende et al. [13]

21. Lines 434 “Previous studies have mixed results on the effect of organic feeds on digestibility” Can you explain what you mean in this sentence?

22. Lines Lines 435-437 “Research by….[28]” please rephrase the whole sentence

23. Line 438 “ Tacon & Metian” please rewrite it as “ Previous studies reported that aquafeeds formulated with alternative protein sources had higher ADCs for protein and energy in various fish species, such as (in which species is it referring to? Please add it)   ….. [8]

24.Lines 458 (Mende et al.[….]) and line  462 ( Monge-Ortiz et al. […]) Please add the reference

25. Line 464 Overall, the findings ….please ad “Overall, the findings from the present study suggest that…

26. Line 491, Please replace as However Fronte et al. [32] investigated the impact of …..

27. Line 495.. please replace as Furthermore, according to Torrecillas et al. [33].

 A general comment on how these results translate into practice - FCR = 1.7, 1.9, 2.05, 2.25,  is not promising. Can you explain in the discussion section, please?

Lines 514-521: In conclusion, can you explain what the present study's results mean in practice?

 Minor editing of the English language required

Author Response

Response to Reviewer 3 Comments

Dear Editor

We have carefully read the reviewers’ comments regarding the manuscript recently submitted to Fishes," New organic raw materials for Gilthead seabream (Sparus aurata) feeding effects on growth, nutritive parameters, digestibility, and histology.” Below we respond to each of the suggestions by the reviewers. The manuscript has been thoroughly revised in response to the reviewers' suggestions. The revised manuscript has been provided to a native English-speaking translator for review. We will re-upload a manuscript that responds to comments and changes and hopes that the revised manuscript will be published in FISHES smoothly.

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Reviewer 3.

Point 1: Throughout your manuscript (abstract, introduction, results, discussion, and conclusion), please where it is written seabream, and replace it with "gilthead seabream."

Response 1: Revised and corrected throughout your manuscript.

Point 2: Line 54, Gambelli et al. [4], please rewrite as According to Gambelli et al. [4] …..and reword the whole sentence

Response 2: Revised and corrected lines 58

Point 3: Line 69-70: A few studies ……., please add a reference. 

Also, in the same Line 69:  Li et al. […] is missing a reference and should be written

or rewritten -rephrase the above sentence on the following lines, line 69 to line 72 (Li et al. [...],...... to growth performance)

Response 3: Revised and corrected line 78.

Point 4: Line 76 Another study by Khieokhajonkhet et al. [the reference is missing] ….,

It is suggested to rewrite it as "Previous studies evaluated the use of ….., and protein utilization [11]

Response 4: Revised and corrected lines 86-88.

Point 5:      Line 92  Another study… please rewrite as "On the other hand, it was reported by  Estevez et al. [3] that, to evaluate new organic ingredients, such as green pea protein and brown seaweed, to replace fish meals in feeds suitable for organic production.

Response 5: Revised and corrected lines 106-88.

Point 6: Line 110-111 "For that purpose." Please rewrite it: " In total, were used 300 fish with an average mean weight of 97 g ± SEM and average mean length ……, and were distributed in 12 tanks of 4000L volume.

Response 6: Revised and corrected lines 126-128.

Point 7: Throughout the M&Ms section, the instruments used must contain all full information (i.e., model, company, city, country).

Response 7: Throughout the M&Ms section, the instruments are revised and contain full information(i.e., model, company, city, country).

Point 8: Line 114-115 “The trial was within a saltwater recirculating system of ….” Please explain. Also, in the same lines you write of 75 m3, please add m3 as m³

In the same lines, the number of individuals that were added in the tanks is unclear. Please add it.

Response 8: Revised and corrected lines 133-134.

Point 9: Line 117-119: “ Additionally,……, and pH.” It would be best to write the water quality ranges for the parameters that you mentioned in the ms (DO, ToC, ammonium, nitrate, nitrite, salinity, and pH) and whether they were kept constant throughout the experiment since you have not included water quality in the results

Response 9: Revised and corrected lines 138-139.

Point 10: Lines 123-127, The replacement was 100%? Please write it.

Response 10: Yes was 100%. Added line 148.

Point 11: Line 124: please delete the space and write it as “ ^oC.”

Response 11: Revised and corrected lines 150.

Point 12: Line 165: Briefly explain why was sampled three fish from each tank..

Response 12: Three fish from each tank to ensure we have representative samples for analysis. Added line 197

Point 13: Line 194-197: “To measure the biometric parametric of the fish,” the fish were anesthetized. Please explain (the anesthetic that was used and the dose)

Response 13: Revised and added to line 229.

Point 13: Line 199-201:Please give us more information in detail about the experiment. Was it a different experiment that was conducted in another RAS? Correct?

Response 13: Yes, correct it was a different experiment conducted in another RAS but in the same laboratory. The digestibility experiment was conducted in the same laboratory but with different tanks with the same conditions as the growth experiment. More information was added in lines 235-137.

Point 14:   . Lines 219 -224,  “Please explain briefly,” Were the fish being sacrificed?

Response 14: It is explained in lines 260-262. The fish were humanely euthanized by immersing them in ice-cold water with a small amount of clove oil as an anesthetic. They were then promptly dissected to obtain the digestive tract.                                                              

Point 15: Table 4 “ESM”  do you mean "SEM"? Please replace it. Also, in Table 4,"final weight" the letters "a, b" indicate significant differences to be rewritten in exponents.

Response 15: Revised and corrected.

Point 16: In Tables 5, 6, 7 8,9, 10. the letters "a, b" indicate significant differences to be rewritten in exponents.

Response 16: Revised and corrected.

Point 17: . Line 358 “table 10”, maybe it's table 9". Please renumber the table and within the text

Response 17: Revised and corrected.

Point 17: Line 377 Table 11” Table 10??? Renumber the table and within the text.

Response 17: Revised and corrected.

Point 18: 18. Figure 2 and Figure 3 provide a better analysis of the graph.

Response 18: Revised and corrected. I hope that the analysis of the figures now is sufficient and better. Line 273, figure 1. Line 382 Figure 2.

Point 19: Lines 414, “In 2017, ….”, please rewrite it as” According to Di Marco […reference ] the organic……..

Response 19: Revised and corrected, line 469.

Point 20: Lines 421 “Mende et al.”  Please rewrite it as Mende et al. [13]

Response 20: Revised and corrected, line 477.

Point 21: Lines 434 “Previous studies have mixed results on the effect of organic feeds on digestibility” Can you explain what you mean in this sentence?

Response 21: The statement implies that previous research on the impact of organic feeds on digestibility has yielded varying outcomes. In other words, some studies have shown the positive effects of organic feeds on digestibility, while others have not found significant improvements. The results of these studies are not consistent or conclusive, suggesting that further investigation is needed to fully understand the relationship between organic feeds and digestibility in the context of fish farming.

Point 22:  Lines 435-437 “Research by….[28]” please rephrase the whole sentence

Response 22: Revised and rephrased, line 493. The study conducted by Amirkolaie et al. [34] revealed that substituting fishmeal with poultry by-product meal (PBM) reduced the digestibility of dry matter, fat, and protein in rainbow trout.

Point 23: Line 438 “ Tacon & Metian” please rewrite it as “ Previous studies reported that aquafeeds formulated with alternative protein sources had higher ADCs for protein and energy in various fish species, such as (in which species is it referring to? Please add it)   ….. [8]

Response 23: Revised and corrected, added species line 499. Previous studies reported that aquafeeds formulated with alternative protein sources had higher ADCs for protein and energy in various fish species, such as Tilapia and Catfish [8].

 Point 24: Lines 458 (Mende et al.[….]) and line  462 ( Monge-Ortiz et al. […]) Please add the reference

Response 24: Revised and corrected lines 521, 526.

Point 25: Line 464 Overall, the findings ….please ad “Overall, the findings from the present study suggest that…

Response 25: Revised and corrected, line 528.

Point 26: Line 491, Please replace as However Fronte et al. [32] investigated the impact of …..

Response 26: Revised and corrected, line 469.

Point 27: Line 495. please replace as Furthermore, according to Torrecillas et al. [33].

Response 27: Revised and corrected, line 562.

A general comment on how these results translate into practice - FCR = 1.7, 1.9, 2.05, 2.25,  is not promising. Can you explain in the discussion section, please?

Response: This paragraph was added to lines 484-488. The FCR values obtained in this study, indicate that fish could be probably fed slightly less maintaining the same growth at the trial temperature. But FCR is consistent with the temperature, growth and feed intake showed.

Lines 514-521: In conclusion, can you explain what the present study's results mean in practice?

 Response: I have change this paragraph, trying to give a more practical conclusion as suggested:. In practice, the results show that TRO IBE and INS meal can be used to replace fishmeal, without harm of the growth performance, nutrient utilization, and intestinal health. Further research and optimization of organic diet formulations may be necessary to improve and maximize efficiency but the organic ingredients have a promising future in the aquaculture of seabream farming. These findings provide valuable information on the effects of different organic diets on digestibility and gilthead seabream liver and intestinal morphology. They could be used to improve sustainable and healthy aquaculture practices

Minor editing of the English language is required.

A native English-speaking teacher has reviewed this manuscript to ensure the integrity of grammar and wording. I hope that the revised manuscript can be successfully published in FISHES.

 To conclude, we thank the reviewers for their comments/suggestions, as they allowed us to improve the manuscript greatly.

 Sincerely yours,

The authors

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have revised the paper according to my comments, so I suggest it will be accepted.

Reviewer 3 Report

Dear Authors

the manuscript has been revised in all highlighted points and can therefore be published in the present form