Dynamics of Fatty Acids in Pikeperch (Sander lucioperca) Larvae and Juveniles during Early Rearing and Weaning in a Commercial RAS—Implications for Dietary Refinement

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

Reply to the Reviewers:

First of all, thank you very much for the time and effort you put into reviewing our manuscript. We appreciate it very much and know that through such an exchange the manuscript is greatly improved.

In addition to the changes in the text mentioned hereafter, other, usually minor, changes were made in the manuscript if we felt they were necessary and improved the manuscript, including language revision.

Reviewer 1:

Abstract:

Question 1:        Line 23 - How many samples?

We included the following sentence into the Abstract in line 24 and 25 (revised version “…, with a total of 6 sampling days, with 4 to 5 replicates per sampling day and between 1 and 25 pikeperch larvae per individual sample, depending on larval biomass.”

Question 2:        What was size of animals?

We included the following sentence into the Abstract in line 25 and 27 (revised version) “The biomass of sampled pikeperch larvae varied from 0.1 to 420 mg (dry mass DM), depending on the age of the larvae, and the initial length of the pikeperch larvae was about 4.5 mm.”

Comment 1:       Dynamics of fatty acids are not shown in the abstract, may be inlided.

Thank you for mentioning this and we agree to the extent that a representation of the dynamics would be helpful, especially since it is mentioned in the title.

Nevertheless, since the sampling period was over 56 days post hatch and a total of 16 fatty acids were included in the table, of which 14 showed dynamics over the sampling period, we did not see a feasible way to incorporate these dynamics into the abstract without confusing the reader with too much information, or unnecessarily complicating the abstract. Besides, the intention of the section in lines 28 to 30 (revised version) was to show the dynamics of fatty acids using total fatty acids.

Question 4:        Line 36-39: We cannot conclude based on review? It therefore, requested to conclude your finding and not the researchers finding.

                            We have added the term "...the offered..." to line 40/41(revised version). Besides that, the statements are based on our own experience during feeding of pikeperch larvae with Brachionus calyciflorus and Brachionus plicatilis, which proved to be a more suitable live feed compared to Artemia in the early stages of development.

Intro:

Comment 2:       Line 65 - Change this line.

Since it was not totally clear which sentence in line 65 (submitted version) was meant by the comment, the two sentences that were in line 65 (submitted version) were changed to the following way. In lines 68 to 72 (revised version).

“Numerous studies have shown that pikeperch larvae can be reared with Artemia [7–14] under aquaculture conditions, but still with unsatisfactory mortality rates. It should thus be investigated whether Artemia can completely satisfy the nutritional requirements of pikeperch larvae.”

Comment 3:       Lines 69-70 - rewrite this sentence.

The sentence was changed to the following version, in line 75 to 81 (revised version).

“Palm and co-workers [16] assumed that high Mono Unsaturated Fatty Acid (MUFA) contents and high levels of eicosapentaenoic acid (20:5(n-3); EPA) in combination with the absence of 22:6(n-3) (DHA) in brine shrimp are unfavorable for the early development of pikeperch. This leads to a high EPA/ARA ratio and especially a low DHA/EPA ratio, which might be possible reasons for high mortality rates in the conventional rearing process of the fry.”

Material and Methods:

Question 5:        Line 108 - What is micro Artemia? May be explained

                            The following explanation was included in line 120 to 123 (revised version).

“Micro Artemia are small Artemia selected by mechanical separation, which can hatch faster. Due to the earlier hatching, the Micro Artemia are also smaller than conventional Artemia and can therefore also be taken up by small and sensitive fish larvae."

Comment 4:       Mention the size of larvae

                            The following sentence was included in line 116 and 117 (revised version).

“Pikeperch larvae had an initial length of 4.5 ± 0.1 mm and an initial biomass of 0.1 ± 0.00 mg (DW). External feeding…”

Comment 5:       Physico-chemical parameters (DO, PH, HARDNESS AND CO2) of RAS is Important? Need to be included in manuscript.

                            Thank you for bringing this to our attention and yes, this data is important. We included the following information in line 112 to 114 (revised version).

                            “The nitrogen compounds were analyzed regularly and were 0.11 ± 0.05 mg*L^-1 for ammonium (NH₄⁺), 0.58 ± 0.84 mg*L^-1 for nitrite (NO₂^-) and 57.72 ± 9.69 mg*L^-1 for nitrate (NO₃^-). The recorded average pH was 8.21 ± 0.09.”

Question 6:        What about larval survival?

Due to the fact that this was not a laboratory experiment, but an accompanying data acquisition in a research hatchery with production scale, no reliable data concerning the mortality rates are available. Each larval tank had a volume of 500 liters and was initially stocked with 100 pikeperch larvae per liter, resulting in a population of 50,000 larvae per tank and there was a total of 8 stocked larval tanks. The recording of reliable mortality rates is a very demanding task for such a scale, which could not be realized with the existing human and time capacities.

Discussion:

Comment 6:      Discussion Section is very lengthy. Reduce the size.

Yes, the discussion is long, but from our point of view it contains necessary information that cause the length. We deliberately did not try to reduce the length of the discussion, as we believe that this would also reduce readability and comprehensibility.

Question 7:        Line 338 - Why the level of LA, ALA and DHA decreases, need to be explained?

                            We added the following sentence in 361 and 362 (revised version).

“This could be due to a reduced concentration or the absence within the microdiets or an increased demand during the early development.”

Comment 7:       Line 349 - Nowhere s mentioned that larvae are initially fed with micro algae? Please mention and name the species.

                            The hatchery does not use microalgae when feeding the pikeperch larvae, and there is no mention of this in the manuscript. While we at the University of Rostock have performed feeding of pikeperch larvae under green water conditions in other experiments and other publications, this was not the case here.

Author Response File: Author Response.pdf

Reviewer 2 Report

This study describes the dynamic of different sources of food from the perspective of fatty acid composition in the early development, weaning, and rearing of pikeperch larvae and juveniles. Although some efforts have been made for describing the use and fate of fatty acids along different growth stages, this study did not achieve its goal and fell short in several respects. First, as neutral (lipid reserves) and polar (membrane lipids) fatty acids were not separated, and were analyzed as total lipids, authors are limited to discussed the results in regard to its use by fish as energy source. On contrary, composition of fatty acids from polar lipids are commonly analyzed to support  all those important physiological changes that occurs during the early fish development and that are described in several published works. Although the fed sources used in this work are commonly used in fish nutrition, is concluded that those fed sources were not adequate for this fish species. Even, the same authors proposed use rotifers instead artemia, also they mentioned some supplements that could or should be used as enrichments in order to improve performance of larval rearing of this fish species.  Also it is mentioned by the authors, that different fish growth stages reflects the fatty acid composition of fed used. The conclusion from all these, is that feds used as fatty acids sources were inadequate or some other sources should be tested. I suggest that efforts should be redirected to understanding the dynamics of fatty acids in reproduction and ontogenetic development in wild fish, in order to later apply this knowledge to the nutrition of larvae and juveniles reared in captivity. I consider that information showed in this study is insufficient and inconclusive. The reported fatty acid composition was only related to a single growth parameter of the fish. No other parameters of rearing performance or physiological aspects of the fish were addressed. For example, what happens with the development of the digestive system of fish, this is normally addressed through the analysis of the tissues by histology. Deficiencies of fatty acids are also reflected in growth of organs and tissues, as eyes, brain, spine, etc.  Finally, I recommend that the author reconsider the rationale and the objectives of this work, but also that the results shown in this study should be complemented with additional analyzes that help to better explain the requirements and use of fatty acids during larval and juvenile culture of this fish species. Therefore, my recommendation is to reject this study for publication in this journal.  

No comments.

Author Response

Reply to the Reviewers:

First of all, thank you very much for the time and effort you put into reviewing our manuscript. We appreciate it very much and know that through such an exchange the manuscript is greatly improved.

In addition to the changes in the text mentioned hereafter, other, usually minor, changes were made in the manuscript if we felt they were necessary and improved the manuscript, including language revision.

Reviewer 2:

First of all, we would like to thank you again for the time invested and regret that we were not able to convince you with our manuscript.

The changes you have suggested are all correct and would definitely improve the manuscript, but this is far beyond the scope of this study and is not practical. Consequently, it will not be possible for us to implement these recommendations for this manuscript with the study described, but we will be happy to consider the recommendations in future studies and experiments.

Author Response File: Author Response.pdf

Reviewer 3 Report

In lines 266, 275 and 537 ARA was given as the abbrevation for 20:4 n-3. Should ARA be 20:4 n-6 as in line 265?

Line 316 It is mentioned that the survival rates are low. But could you corroborate that by the dana?

In lines 266, 275 and 537 ARA was given as the abbrevation for 20:4 n-3. Should ARA be 20:4 n-6 as in line 265?

Line 316 It is mentioned that the survival rates are low. But could you corroborate that by the data?

Author Response

Reply to the Reviewers:

First of all, thank you very much for the time and effort you put into reviewing our manuscript. We appreciate it very much and know that through such an exchange the manuscript is greatly improved.

In addition to the changes in the text mentioned hereafter, other, usually minor, changes were made in the manuscript if we felt they were necessary and improved the manuscript, including language revision.

Reviewer 3:

Question 1:        In lines 266, 275 and 537 ARA was given as the abbreviation for 20:4 n-3. Should ARA be 20:4 n-6 as in line 265?

                            Thanks for bringing this to our attention and yes you are right ARA should be written as 20:4(n-6). We have changed it accordingly in the lines 287, 296 and 562 (revised version).

Question 2:        Line 316 It is mentioned that the survival rates are low. But could you corroborate that by the data?

                            We included additional data from the mentioned literature in the lines 335 to 338, see the additions below:

                            “The survival rates of 7.9 ± 3.5% per tank described by Colchen et al. (2020) [22] and 11.3% by Péter et al. (2023) [23] were rather low survival rates compared to the survival rates ranging from 64.0 to 93.7% by Ballesteros et al. (2023) [24] and up to 94.0% by Bischoff and Kubitz et al. (2022) [25].”

Author Response File: Author Response.pdf

Reviewer 4 Report

GENERAL conclusion on the manuscript

 This study is dealing on the first place with assessment of fatty acid profile of larvae and fry of pike perch during 56 days of weaning period using the feeding strategy commonly applied at fish farms. Unfortunately, behind the fatty acid level determined in the fish only some growth parameters are assessed. Overall, the manuscript would be interesting if some physiological impact of diets will be evaluated. For example, mortalities, effect of stress or deformities etc.

There are some conclusions that are not supported by the results of this study. E.g. in the Abstract, Line 33: “A temporary lack of essential fatty acids cause dysfunctions and eventually mortalities”. Where this statement coming from?

It is mentioned that fatty acid profile in fish is reflecting dietary composition, as a statment (Line 81). In this sense the results presented in the frame of this study do not have novelty and also the hypotheses raised were already demonstrated. As conclusion, the aim of this study is not well defined. Moreover, the fatty acid profile and specific growth rate data are not supporting the main conclusions of these feeding experiment. (”first feeds was apparently insufficient, which caused dysfunctions and mortalities in pike perch larvae and juveniles”)

SPECIFIC comments:

Line 179 Specific growth rate is calculated between the sampling dates. According to this in Line 133 t=0 is not correct. should be t= initial and Wi

Line 193 TFA is not defined.

The Results and Discussion parts are very lengthy, unnecessary to write in detailed if the data are presented in the table.

Line 376, 431 There are statement related to the level of fatty acid in eggs or diet and reproduction success. I am warning to make any relationship between the maternal nutrition and larvae nutrition in sense of the role of fatty acids. I suggest to delete these sentences.  

Comments for author File: Comments.pdf

Author Response

As already written for Reviewer 2 regarding mortality, we have attempted to address the comment regarding other relevant parameters and have added this in an introductory text to the Material and Methods section in lines 94 to 106 of the newly revised manuscript.

“For a comprehensive representative and reproducible analysis of the effects of live feed on the development of pikeperch larvae and juveniles, further parameters such as mortality, deformations or the effects of stress would be helpful and necessary in addition to the fatty acid and growth data (specific growth rate SGR), which were used here. However, due to limited financial and personnel capacities, not all these parameters could be determined in the course of this experiment. This is also due to the fact that the thematic priorities of the Institute for Fisheries of the State Research Centre for Agriculture and Fishery Mecklenburg-Western Pomerania (LFA MV) in Hohen Wangelin at that time were the investigation of breeding and hatching rates as well as growth parameters but not yet the above-mentioned parameters.

These parameters have already been collected for subsequent experiments and will now also be collected for future experiments, as long as this is appropriate for the thematic priority of the research.”

The statement in the abstract lines 37 and 38 was already changed in the first revision to the sentence " A temporary lack of essential fatty acids can cause dysfunctions and eventually mortalities in pikeperch larvae and juveniles.", which refers to the possibility of dysfunctions and mortalities, but does not declare them as general.

The objective of the paper was correctly stated that the fatty acid profile in fish reflects dietary composition (line 86 to 92) and it is also true that the results presented in this study are not completely new and also the hypotheses put forward have already been proven, but we have only a few publications that have been carried out in a research facility on a commercial scale, i.e. the available publications are almost all based on lab scale experiments. Therefore, we consider the data and findings presented here as worthy of publication and assume a professional benefit for the readership.

However, the statement that the data are not supporting the conclusion “first feeds was apparently insufficient, which might have caused dysfunctions and mortalities in pikeperch larvae and juveniles.” Has to be examined more specifically.

On the one hand, an important part of the sentence was omitted or neglected for the statement on the conclusions presented “As conclusion, the aim of this study is not well defined. moreover, the fatty acid profile and specific growth rate data are not supporting the main conclusions of these feeding experiment. (”first feeds was apparently insufficient, which caused dysfunctions and mortalities in pikeperch larvae and juveniles”)”, since the supply of individual PUFA was explicitly addressed here. On the other hand, we have changed the statement and added the terms "might have" to the sentence in line 600.

Thank you for the hint and the correct statement that the SGR were determined for the different intervals. We have changed the formula and the associated explanations in the lines 163 to 165.

Thank you very much for the comment. We have tried to include the definition of the term TFA = total fatty acids in the heading of Table 2, even if we had already defined TFA in the abstract in line 28, but of course it could be that the definition is not noticed here.

Concerning the length of the Results and Discussion, we tried to shorten the manuscript by trying to avoid possible duplications in the text and tables.

Thank you for your assessment. The intention of the sentence (now in lines 384 to 386) is to discuss the effects or benefits of Micro Artemia during the first few days of life of pikeperch larvae and no statement should be made about the relationship between maternal nutrition and reproductive success. In general, we believe that the maternal nutrition ideally reflects the best possible nutrient composition in the eggs and consequently the newly hatched larvae, provided the females were healthy and had an adequate food supply and were able to pass the nutrients to the eggs.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I reviewed the responses issued by the authors to the comments of the reviewers, including those presented by myself. My opinion about this study remains the same. The composition of fatty acids of the diets, were not selected in relation to the nutritional requirements for this species of fish, obtaining poor results in the growth and culture of the fish. The same authors acknowledge that the diets were inadequate. In general, the focus of the work is inadequate, many other things could have been implemented as I mentioned in the previous review. I continue suggest not accepting this study for publication.

Author Response

Thank you again and yes, we were not able to change your mind because as addressed in the first revision, this was not the focus of the original experiment and this would have been far beyond the scope of the revision.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript has been improved. The survival rate is still not known and should be measured in future studies with similar aim. 

Author Response

Thank you again for reviewing the manuscript again.

We have tried to address the comment regarding mortality and have added this to an introductory text to the Material and Methods section in lines 94 to 106 of the newly revised manuscript.

“For a comprehensive representative and reproducible analysis of the effects of live feed on the development of pikeperch larvae and juveniles, further parameters such as mortality, deformations or the effects of stress would be helpful and necessary in addition to the fatty acid and growth data (specific growth rate SGR), which were used here. However, due to limited financial and personnel capacities, not all these parameters could be determined in the course of this experiment. This is also due to the fact that the thematic priorities of the Institute for Fisheries of the State Research Centre for Agriculture and Fishery Mecklenburg-Western Pomerania (LFA MV) in Hohen Wangelin at that time were the investigation of breeding and hatching rates as well as growth parameters but not yet the above-mentioned parameters.

These parameters have already been collected for subsequent experiments and will now also be collected for future experiments, as long as this is appropriate for the thematic priority of the research.”

Furthermore, in the Conclusion section, we reiterated the future inclusion of data on mortality, deformities, and effects of stress in line 601 to 602.

“In the future, more efforts should be made to incorporate parameters such as mortality, deformities, and the effects of stress into data collection.”

Author Response File: Author Response.pdf

Reviewer 4 Report

The authors answered my question and modified according to my suggestion. The explanation given for the missing parameters is acceptable on my side.

Author Response

Dear Reviewer,
Thank you very much for the repeated review of the manuscript and the positive evaluation. We appreciate your work very much and thank you for the opportunity to improve our manuscript with your help.