Adult European Seabass (Dicentrarchus labrax) Perform Well on Alternative Circular-Economy-Driven Feed Formulations

Round 1

Reviewer 1 Report

Adult European seabass (Dicentrarchus labrax) perform well on alternative circular-economy-driven feed formulation (Sustainability-1724179)

The above MS was conducted to evaluate the effect of alternative feed formulations on health status, welfare parameters, sensory attributes, growth performance and nutrient digestibility in European seabass (Dicentrarchus labrax) over an 83-day feeding trial. The topic is quite interesting for aquaculture; however, there are some issues arising through the MS.

Although the authors gave procedures for nutrient digestibility and sensory attributes, they did not provide any results accordingly.

The authors substitute the dietary protein resources but they never talk about amino acid profile in the diets.

Statistical differences were expressed in a confusing way, not corresponding with multiple comparison aspect. For example, the authors provide 2 P values for 1 parameter in several cases which is not consistent with multiple comparison on the means.

The superscript letters for statistical differences seem confusing in almost all cases; therefore, they need to be critically revised. The letters also should be used in order with the increasing or decreasing values.

The results section is poorly prepared and written; therefore, needs to be re-written, giving the findings and statistical differences clearly.

Even though the initial and final weights are similar, then how come the growths are different?

The discussion is very narrow and out of focus, thus it needs to be enriched and re-organized after a critical revision.   

Below are some other issues:

L15-17: This study provides results for the effect of alternative feed formulations on health status, welfare parameters, sensory analysis and growth performance in European seabass (Dicentrarchus labrax) over an 83-day feeding trial.

L27-28: not clear

L32: give the who name for the FAO when using as the first time

L52: delete “most”. The sentence reads as “FM is considered…..”

L93: % instead of per cent

L98-99: not clear

L106-107: not clear

L111-113: re-formulate the sentence

L130: feeding experiment

L131: starting the 83-day feeding trial..

L148: ad libitum will be italic

L149: “thirty” instead of “30”

L161: (Table 2)

L164: here and anywhere else use the abbreviation appropriately

L189: do not compare the proximate composition of the diet

L214: was the supernatant removed?

L256: Table

L271: performed according to

L273: “thirty” instead of “30”

L303: what is this relative growth why not specific growth rate

L352: here any anywhere else “(P<0.05)” or “(P=0.035)”. However, although your P value for weight gain is 0.035 according to the corresponding table, you are coming up with two different P values which is not understandable when you do multiple comparison. Just give the results that you have in the table in an appropriate way.

L372-373: same as above

L387-395: I do not see any statistical differences in Table 8!

L406-407: re-formulate the sentence

L422-424: same as L352

L435-436: same as above

L445: Where are the results?

L462: delete the Latin name

L473: feed efficiency

L474: differences were found

L501-502: re-formulate the sentence

L503: the difference is not a big deal in your case

L507: check the data!

L535: replacement

L541: When latter effect is larger, the availability…..

L553: Among others, plasma metabolites like glucose and total protein are considered….

L557: was replaced

L558: decrease in

L583: acid

L584: oleic… linoleic

L591: is replaced

L594-595: re-formulate

L598-600: re-formulate

 

 

 

 

Author Response

Dear Reviewer 1,

Thank you very much for your conscientious study of our manuscript and your valuable comments. Your input helped us to improve the manuscript significantly. We hope that we have adequately responded to your comments, which are addressed as follows:

Point 1: Although the authors gave procedures for nutrient digestibility and sensory attributes, they did not provide any results accordingly.

Response 1: The results for the sensory attributes were statistically not significantly different, so the authors decided for clarity reasons to exclude the exact numbers in the result section (Line 517). For the nutrient digestibility, some technical problems occurred during the analysis: analysing the carcass of the different fed fish for protein and fat was unfortunately impossible. Therefore could, for protein and fat, only the ADC of the feed be included. (see Table 3, Line 377).

Point 2: The authors substitute the dietary protein resources but they never talk about amino acid profile in the diets.

Response 2: The amino acid profile was not conducted for the different feeds due to limiting capacities of the PhD student. Analysing the profile is very costly and time-consuming and could not be done at the time of this study. For subsequent studies, we will concider this type of analysis. However, we decided that the overall protein content is sufficient to compare feed as we do not expect to see significant differences in the amino acid profile. Furthermore, were the feed concepts in a way formulated that ensured all nutritional requirements for the seabass and the essential amino acids were supplemented.

Point 3: Statistical differences were expressed in a confusing way, not corresponding with multiple comparison aspect. For example, the authors provide 2 P values for 1 parameter in several cases which is not consistent with multiple comparison on the means. The superscript letters for statistical differences seem confusing in almost all cases; therefore, they need to be critically revised. The letters also should be used in order with the increasing or decreasing values.

Response 3: The p.values in the tables were constantly referring to a ONE-Way ANOVA analysis, while the p-values in the text referred to the post hoc test to give more insights into the statistical difference between the different diets. We admit that the post hoc test is confusing, and we changed/deleted the numbers accordingly. We also revised the superscript letters in the manuscript according to increasing values.

Point 4: The results section is poorly prepared and written; therefore, needs to be re-written, giving the findings and statistical differences clearly.

Response 4: The result chapter was adjusted in the manuscript accordingly.

Point 5: Even though the initial and final weights are similar, then how come the growths are different?

Response 5: The initial and final weights were overall quite similar, that is true. Nevertheless, did the fish of the different treatments show growth differences due to high individuality. Thanks to our tagging procedure we could identify these differences and a change in growth between treatments could be identified (Line 549-553).

Point 6: The discussion is very narrow and out of focus, thus it needs to be enriched and re-organized after a critical revision.   

Response 6: amended in the manuscript.

Below are some other issues:

Point 7: L15-17: This study provides results for the effect of alternative feed formulations on health status, welfare parameters, sensory analysis and growth performance in European seabass (Dicentrarchus labrax) over an 83-day feeding trial.

Response 7: amended in the manuscript. Line 15

Point 8: L27-28: not clear was adjusted etc.

Response 8: amended in the manuscript. Line 25-28

Point 9: L32: give the who name for the FAO when using as the first time

Response 9: amended in the manuscript. Line 32

Point 10: L52: delete “most”. The sentence reads as “FM is considered…..”

Response 10: amended in the manuscript. Line 53

Point 11: L93: % instead of per cent

Response 11: amended in the manuscript. Line 96

Point 12: L98-99: not clear

Response 12: amended in the manuscript. Line 99-103

Point 13: L106-107: not clear

Response 13: amended in the manuscript. Line 110-113

Point 14: L111-113: re-formulate the sentence

Response 14: amended in the manuscript. Line 116

Point 15: L130: feeding experiment

Response 15: amended in the manuscript. Line 136

Point 16: L131: starting the 83-day feeding trial..

Response 16: amended in the manuscript. Line 138

Point 17: L148: ad libitum will be italic

Response 17: amended in the manuscript. Line 157

Point 18: L149: “thirty” instead of “30”

Response 18: amended in the manuscript. Line 159

Point 19: L161: (Table 2)

Response 19: amended in the manuscript. Line 171

Point 20: L164: here and anywhere else use the abbreviation appropriately

Response 20: amended in the manuscript. Line 170

Point 21: L189: do not compare the proximate composition of the diet

Response 21: We don’t understand this comment. Could you please specify the question.

Point 22: L214: was the supernatant removed?

Response 22: amended in the manuscript. Line 225

Point 23: L256: Table

Response 23: amended in the manuscript. 382

Point 24: L271: performed according to

Response 24: amended in the manuscript.

Point 25: L273: “thirty” instead of “30”

Response 25: amended in the manuscript.

Point 26: L303: what is this relative growth why not specific growth rate

 

Response 26: From our perspective we understand the relative growth rate (RGR) is mathematically based on the absolute growth rate. It displays the absolute increase in relation to the initial weight/length and is reported as a percentage increase over time. The specific growth rate is defined as the rate of increase of biomass. According to the literature, specific and relative growth rates provide similar overall results, but relative growth rates give a more precise long term overview. (Crane et al., 2019).

Point 27: L352: here any anywhere else “(P<0.05)” or “(P=0.035)”. However, although your P value for weight gain is 0.035 according to the corresponding table, you are coming up with two different P values which is not understandable when you do multiple comparison. Just give the results that you have in the table in an appropriate way.

Response 27: changed in all sections of the paper. The second p-values were from the post hoc tests, but you are right and they do not apply in our analysis.

Point 28: L372-373: same as above

Response 28: amended in the manuscript.

Point 29: L387-395: I do not see any statistical differences in Table 8!

Response 29: amended in the manuscript. Line 447

Point 30: L406-407: re-formulate the sentence

Response 30: amended in the manuscript. Line 452

Point 31: L422-424: same as L352

Response 31: amended in the manuscript.

Point 32: L435-436: same as above

Response 32: amended in the manuscript.

We are unclear if the row information below is correct, as the issues do not match the rows. We would have liked to change it but were unsure what exactly was meant.

Point 33: L445: Where are the results?

Response 33: This comment is confusing. The plasma results are all seen in Table 10, right below the text. Please indicate which results are exactly missing, thank you. Line 493

Point 34: L462: delete the Latin name

Response 34: We can not see a Latin name in these lines or close lines. Please state again, which Latin name you mean.

Point 35: L473: feed efficiency

Response 35: amended in the manuscript. Line 532

Point 36: L474: differences were found

Response 36: amended in the manuscript. Line 545

Point 37: L501-502: re-formulate the sentence

Response 37: amended in the manuscript. Line 549 - 553

Point 38: L503: the difference is not a big deal in your case

Response 38: We double checked the data and understand that the difference overall may not be too big, but it´s still significantly different and should be mentioned in the manuscript. Line : 557

Point 39: L507: check the data!

Response 39: amended in the manuscript. Line 561

Point 40: L535: replacement

Response 40: amended in the manuscript. Line 596

Point 41: L541: When latter effect is larger, the availability…..

Response 41: amended in the manuscript. Line 623

Point 42: L553: Among others, plasma metabolites like glucose and total protein are considered….

Response 42: amended in the manuscript. Line 635

Point 43: L557: was replaced

Response 43: amended in the manuscript. Line 639

Point 44: L558: decrease in

Response 44: amended in the manuscript. Line 640

Point 45: L583: acid

Response 45: amended in the manuscript. Line 662

Point 46: L584: oleic… linoleic

Response 46: amended in the manuscript. Line 666

Point 47: L591: is replaced

Response 47: amended in the manuscript. Line 675

Point 48: L594-595: re-formulate

Response 48: amended in the manuscript. Line 678

Point 49: L598-600: re-formulate

Response 49: amended in the manuscript. Line 684

Author Response File: Author Response.pdf

Reviewer 2 Report

Despite technological and scientific advances, we see the number of hungry people increase every year in the world. An alternative to meet this demand for foods with high nutritional value is aquaculture. But this aquaculture production has to be more environmentally friendly, so as not to worsen the health of the planet.

For these and other reasons, the manuscript presents very interesting results on the cultivation based on the circular economy of a species with great commercial appeal.

However, some topics of methodology and results caught my attention. which made me indicate a need for a major revision of the article.       

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 2,

Thank you very much for your conscientious study of our manuscript and your valuable comments. Your input helped us to improve the manuscript significantly. We hope that we have adequately responded to your comments, which are addressed as follows:

 

2.2. Major comments

Point 1: How many tanks were used throughout the experiment? In item 2.1, mention 15 tanks. However, in lines 136 and 137, the authors write "36 individual holding tanks". I would like the authors to better explain the experimental design used.

Response 1: For this experiment were 15 out of the 36 tanks used to ensure a density comparable to aquaculture facilities. The rest of the tanks were used as holding tanks for the spare fish. Line 143-145

 

Point 2: Various analyses were made in external laboratories. However, the authors should put as methodologies used.

Response 2: The missing methods were added in the text.

• Line 279- 281
• Line 311-319

 

Point 3: Lines 242 – 244. For all as a raw protein sample was a total protein concentration estimated by the total conversion factor? This was not clear. In addition, as ingredients from different sources were used, care should be taken with the use of this conversion factor value, as it is an average value for meats. Vegetable sources have another conversion value.

Response 3: That is a very good point which we will concider in our next experiment. Here, we used the conversion factor of 6.25 for all samples. In order to analysze the exact conversion factor for a dietal concept there is a lot work involved and theretically the exact factor could be estimated but practically you never exactly know what the fish absorbed. We therefore estimated the conversion factor for this first attempt of feed concepts. We further discussed this in the discussion chapter (Line 581-585).

 

Point 4: Tables 1, 3 and 4 are not results? Furthermore, do the water quality values not vary between the treatments tested?

Response 4: These tables give the baseline data before the experiment. You are right with Tables 3 and 4, and we shift them to the result chapter. The water quality did not differ between the treatments as all tanks were part of the same recirculating system. Therefore, all tanks get the exact water qualities. In addition, we randomly checked the water values inside some of the treatment tanks, and the values were the same and not influenced by the different feed types.

 

 

 

Point 5: Many results showed large standard deviations, very close to the mean values (weight gain in Table 6, for example). Which may have influenced the statistical results. Are these values correct? if so, I believe that an analysis of the individuals can be carried out, since it was possible to identify each fish using the tag.

Response 5: The values are all correct and have been checked several times. We used a practical approach in the analysis suitable for aquaculture facilities. Fish are not measured individually in commercial aquaculture farms, only total growth. Therefore, we decided to use the same practical approach in this work and did not perform an individual analysis.

 

Point 6: Table 11. I believe it would be interesting to carry out the analysis of the same fatty acids in the rations. For us to know, if associated with oleic acid it is just the differences in the food ingested, or is it due to some different metabolic pathway being activated.

Response 6: We agree that this would be an excellent addition to the experiment but can unfortunately not be carried out anymore due to technical difficulties. But we will include this point in our subsequent investigation.

 

Point 7: I understand that alternative sources of fatty acids and proteins should be sought. But are they profitable? I suggest doing a cost analysis of the feed used.

Response 7: a section for the cost analysis was added in lines 705-715

 

2.3. Minor comments

Point 8: What is the life stage (age) of the fish used in the experiment?

Response 8: The fish were approximately 2.5 years old.

 

Point 9: Table 2. Why were these ingredients used? Are there any scientific criteria or were they commercially available?

Response 9: The ingredients were chosen primarily to mimic a commercial feed. The goal was to use commercially available materials as far as possible and potential ingredients from the circular economy to create a feed concept that is more sustainable while not compromising growth.

 

Point 10: In topic 2.3 it is written that there was a collection on the 57th. But this is no longer mentioned throughout the manuscript and no data from this collection is presented. I believe it was a typo.

Response 10: This was not a typo. We included an intermediate sampling to save some data. But this data was never used. You are right; this is confusing and was removed from the manuscript.

 

Point 11: Lines 198-199. Was the baseline collection performed before or after acclimatization?

Response 11: The baseline data was collected after acclimatization. This sentence was added in the manuscript. (Line 205).

 

 

 

Point 12: Were those collected during the experiment gathered? Transforming a single composite sample per tank?

Response 12: The baseline data was obtained from a special tank before the actual experiment. Therefore, we set some fish aside not to disturb the experimental fish in the 15 tanks. All samples of the baseline measurement came from the same tank. Due to the fact that all tanks were connected in a recirculating system, all tanks were identical and the water treatment was the same.

 

Point 13: Is freeze drying an AOAC validated process?

Response 13: Yes, that is the case ( e.g. AOAC 2001.12 (I) ) https://wenku.baidu.com/view/e08a4afb941ea76e58fa0473.html?re=view

 

Point 14: Why was a mineral analysis performed in duplicate?

Response 14: Mineral analysis was performed in duplicates due to a time-intensive measuring technique, which is also very cost intensive. Of course, we know that a duplicate measurement is statistically not the best variant. In addition, we had the problem that during the pandemic the laboratories were closed for more than a year and after the release the workload of the laboratories was quite high and therefore we only got short time windows. We made the best of it and therefore had to opt for the duplicate analysis.

 

Point 15: Table 4. Put as Variations (Standard Deviation or Reflection Calculation).        

Response 15: The same problem arises when measuring the mineral composition in the feed. Here, the time and availability of the laboratory was so limited for the reasons described above that we had no other choice and limited ourselves to a single measurement.

Author Response File: Author Response.pdf

Reviewer 3 Report

The current study discusses an important point in the field of aquaculture challenges. But the experimental diets seem like concepts not rather than experimented diets to compare. All aquaculturists looking for alternative protein sources. But here, it's difficult to judge the experimented diets due to extensive complexity. While, on the other hand, the authors did not separate and/or compare each category's source of alternative protein sources. So, it's too difficult to judge or say that diet no. (X) is recommended to use in aquaculture, in general. This manuscript is presented well with high efforts but unfortunately, I am so sorry to say that I can not accept this manuscript for publication due to the above problems in the methodology and the experimental diets.

Author Response

Dear Reviewer 3,

Thank you for your conscientious review of our manuscript and your comments. We regret your decision regarding our manuscript but understand your concerns. With the help of your comments and those of the other reviewers, the manuscript has been thoroughly revised and improved once again. 
Thank you for your efforts and suggestions. We hope the new document can meet the high standards. 

Reviewer 4 Report

I found this paper to be very interesting and the concept is very much needed to sustainably develop aquaculture. There are some issues that I have indicated in the presentation of the manuscript that need attention. The main things for me are the phrasing of some of the sentences, I have offered suggestions in some cases, the reporting of results in the discussion and the lack of proper integration with the current literature in the discussion. I hope you find my comments helpful and thank you for submitting an interesting manuscript.

Line 27 – Should this read like the experimental diets tested here instead of like the here testes experimental diets.

Line 51 – Need to be augmented with instead of by

Line 63 – The language used here is inappropriate and conversational. The sentences does not read well. This is especially important in nowadays situation. Maybe this is especially important in the current situation.

Line 67 – Is this figure for general animal use or just fish? Please clarify for context.

Line 74 – This needs and is known

Line 78 – Animals with simple stomach structures, such as fish. I am not sure I follow this line of thought. What about herbivorous fish? You are referring here to carnivores/piscivores? You almost note this with the next sentence about carnivorous fish.

Line 81 – Please stop using the word nowadays. This is very conversational.

Line 85 – Remove a consequent.

Line 87 – This sentence feels repeated from line 79.

Line 116 – This is methods and should be reported in that section.

Line 128 – kept in the RAS.

Line 134 – Change candidates to individuals.

Line 134 – The mean was around? It was 320.8 ±72.4 g.

Line 136 – Can you please clarify the set up. You note 15 tanks were used. Have you noted the 36 tanks to address the issue of water use without load? If so please clarify.

Line 156 – Just control, not control one.

Line 159 – Were all diets nutritionally complete for this species? If not did you need/have a licence to do the work?

Line 210 – Could you clearly separate uneaten food from faeces?

Line 352 – Where is the 0.002 represented on the table?

Line 352 – You have P-values that are not represented anywhere. Can you rectify this? This happens throughout.

Line 460 – whether instead of if.

Line 473 – have other similar studies shown the same trends?

Line 476 – You don’t have to report the results again. We have seen these previously in the results section. Just the trend is sufficient.

Line 479 – I would like to see more integration with the literature throughout. The discussion feels a little like a list of findings and it would read better if it was more integrated.

 

 

Author Response

Dear Reviewer 4,

Thank you very much for your conscientious study of our manuscript and your valuable comments. Your input helped us to improve the manuscript significantly. We hope that we have adequately responded to your comments, which are addressed as follows:

 

Point 1: Line 27 – Should this read like the experimental diets tested here instead of like the here testes experimental diets.

Response 1: amended in the manuscript. Line 27

 

Point 2: Line 51 – Need to be augmented with instead of by

 

Response 2: amended in the manuscript. Line 52

Point 3: Line 63 – The language used here is inappropriate and conversational. The sentences does not read well. This is especially important in nowadays situation. Maybe this is especially important in the current situation.

Response 3: amended in the manuscript. Line 64

Point 4: Line 67 – Is this figure for general animal use or just fish? Please clarify for context.

Response 4: amended in the manuscript. Line 69

Point 5: Line 74 – This needs and is known

Response 5: amended in the manuscript. Line 77

Point 6: Line 78 – Animals with simple stomach structures, such as fish. I am not sure I follow this line of thought. What about herbivorous fish? You are referring here to carnivores/piscivores? You almost note this with the next sentence about carnivorous fish.

Response 6: amended in the manuscript and added with carnivorous. You are right. This part is only looking at carnivorous fish and not herbivores. Line 81

Point 7: Line 81 – Please stop using the word nowadays. This is very conversational.

Response 7: amended all over the manuscript. Line 83

Point 8: Line 85 – Remove a consequent.

Response 8: amended in the manuscript. Line 88

Point 9: Line 87 – This sentence feels repeated from line 79.

Response 9: amended in the manuscript. Line 91

Point 10: Line 116 – This is methods and should be reported in that section.

Response 10: amended in the manuscript.

Point 11: Line 128 – kept in the RAS.

Response 11: amended in the manuscript. Line 134

Point 12: Line 134 – Change candidates to individuals.

Response 12: amended in the manuscript. Line 141

Point 13: Line 134 – The mean was around? It was 320.8 ±72.4 g.

Response 13: amended in the manuscript. Line 141

Point 14: Line 136 – Can you please clarify the set up. You note 15 tanks were used. Have you noted the 36 tanks to address the issue of water use without load? If so please clarify.

Response 14: clearified in the manuscript. Line 146

Point 15: Line 156 – Just control, not control one.

Response 15: amended in the manuscript. Line 167

Point 16: Line 159 – Were all diets nutritionally complete for this species? If not did you need/have a licence to do the work?

Response 16: Yes, all diets were nutritionally complete for the respective specie. We exchanged the less sustainably produced ingredients with more sustainably produced alternatives and identified that the requirements, such as proteins and energy, were met. We have an animal trial permission for the experiments mentioned in this manuscript (see ethics statement, Line 752).

Point 17: Line 210 – Could you clearly separate uneaten food from faeces?

Response 17: Yes. The faeces sank to the bottom while the uneaten food floated at the top. The feed pellets were netted from the tank after the afternoon feeding, and in a little test before the experiment, we saw that even after one full day, the pellets were still afloat. So even when a pellet was overseen in the afternoon, it was either eaten the next day or still afloat and could be removed in the morning.

 

Point 18: Line 352 – Where is the 0.002 represented on the table?

Response 18: The p.values in the text were referring to a post hoc test. I admit that this was very confusing throughout the manuscript and was removed for clarity.

Point 19: Line 352 – You have P-values that are not represented anywhere. Can you rectify this? This happens throughout.

Response 19: see above

Point 20: Line 460 – whether instead of if.

Response 20: amended in the manuscript. Line 531

Point 21: Line 473 – have other similar studies shown the same trends?

Response 21: Yes, the literature referring is in line 538.

Point 22: Line 476 – You don’t have to report the results again. We have seen these previously in the results section. Just the trend is sufficient.

Response 22: I choose to highlight the most important results in the discussion chapter to emphasize the differences. In our opinion does this clearify the trend more obvious.

Point 23: Line 479 – I would like to see more integration with the literature throughout. The discussion feels a little like a list of findings and it would read better if it was more integrated.

Response 23: amended in the manuscript.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors answered all the questions and doubts of the first review. Taking into account the difficulties of developing the work due to the social isolation necessary to contain the spread of COVID-19, I understand that the manuscript can be accepted in this form.

Reviewer 3 Report

The authors have satisfactorily addressed the reviewer comments.