The Application of Single-Cell Ingredients in Aquaculture Feeds—A Review
Round 1
Reviewer 1 Report
This review is dealing with a very actual and constantly evolving industry. Single cell ingredients, despite very promising characteristics, require further research and strategies for larger-scale and lower cost production as correctly pointed out by the authors.
Scientific literature reviewed is vast and very updated.
Only few minor comments are proposed:
- there is no accordance among the number and titles of the seven steps presented in the introduction and the headings and order of presentation of the sections later in the paper.
- Figure 2 is specific for tilapia protein intake and growth rate relationship. Such information only for this species seems not necessary and does not give further insight to the review. It could be discussed in the text without showing the picture.
Author Response
Response to Reviewers - FISHES-838508
Reviewer 1
4/5 - Is the work a significant contribution to the field?
4/5 - Is the work well organized and comprehensively described?
5/5 - Is the work scientifically sound and not misleading?
5/5 - Are there appropriate and adequate references to related and previous work?
3/5 - Is the English used correct and readable?
This review is dealing with a very actual and constantly evolving industry. Single cell ingredients, despite very promising characteristics, require further research and strategies for larger-scale and lower cost production as correctly pointed out by the authors.
Scientific literature reviewed is vast and very updated.
Only few minor comments are proposed:
Q1.1 - there is no accordance among the number and titles of the seven steps presented in the introduction and the headings and order of presentation of the sections later in the paper.
A1.1 We don’t beleive that this is correct. The review is structured in sections directly reflecting those seven steps, except that because of the volume of data around step 4, we broke that section into subsections based on the different classes of SCI and different aquaculture species classes. Other than that, the review very much follows the sequence of characterization-digestibility-(palatability+utilisation)-Immunological-Processing-Quality. It was difficult to separate out the palatability and utilisation studies, so we combined those two steps in a single (large) section.
Q1.2 - Figure 2 is specific for tilapia protein intake and growth rate relationship. Such information only for this species seems not necessary and does not give further insight to the review. It could be discussed in the text without showing the picture.
A1.2 We disagree, as the relationship shows an important feature of the value of different microalgal products and how variation in the intake x digestibility story combines to affect their value.
Reviewer 2 Report
The review of Glencross is a relatively comprehensive update on the use of single cell ingredients as protein and lipid materials in fish feeding. It is relevant for fish and shrimp nutrition research as there is a growing interest for these ingredients in the context of the development of sustainable aquafeeds. It is well adapted to the scope of Fishes journal.
The authors considered the main groups of Single cell ingredients and their two main utilisation as protein ingredients and oil ingredients in aquafeed. The review is well adapted to fish feeding and nutrition with different chapters and illustrations related to the main crucial points for including such ingredients in formulation. There is an in-depth analysis of the litterature on utilisation of SCI in the main groups of farmed species salmonids, tilapia, marine species and shrimp. Thus it could be published in Fishes.
There is however a recent review in this field also focused on single cell ingredients in aquaculture Jones et al 2020 Recent advances in single cell protein use as a feed. Current Opinion Biotech. The two reviews are complementary but the authors have to include this review in their analysis, to explain the complementarity and to use as far as possible the information given in this review.
The authors did not considered important points for formulation that could be sum up in a specific chapter or in the conclusion : i) the current sources and the costs of these ingredients. It could be useful to have a range of cost for each ingredients compared to fish meal, soya bean meal for SCP and to fish oil and vegetable oil for SCO ii) the pro and the cons of including SCI in aquafeed
Scope of the review :
The classification of single cells organism as microalgae, fungi (yeast) and bacteria does not consider a separate protists group that comprise for instance schizochitrium
It is not clear if the biofloc group includes the mixtures of microalgae produced on CO2 capture from industrial wastes. There are now numerous reviews on these technologies and some of these comprise the characteristics of bioflocs.
Characterisation of SCI
The characterization is only limited to groups up to species but it has to include known strains varieties or lines and their purity level. For instance selected lines start to be used for Spirulina which is reared since a long term.
The potential variability of the characteristics related to rearing conditions of SCI anaerobic or aerobic, bioreactor vs open-field is not considered
Digestibility
The chapter did not considered the effect of SCI on the microbiote and the contribution of microbiote to the known positive effects of SCI
Digestibility of SCP in Mustela vison can be cited in the text but not could not be included in the table.
Processing effect
This is only a simple attempt to review of the effect of SCI processing and on the expected quality of SCI for formulation : availability, requirement, control, management. This chapter could be beneficially enlarged.
Health and immunity
The chapter is only focused on positive effects of SCI. There are however negative effects. For instance micro-algae or cyanophycae can synthetised toxins depending on condition of rearing.
l279-287 Why not including in this chapter polysaccharides mannan & glucan of yeast that also stimulate immune system. This is only mentionned in the general presentation of each group of SCI.
Next steps The scope of this chapter is limited.
Why not mentionning the need for research on the rôle of microbiote, the effect of specific SCI substrates ex poly-hydroxy-butyrate …
This chapter considered only a part of the environmental impact of the production of SCI. The ingredients are produced in various condition from landless pilot plants up to openfield. The environmental impact of plants is not inconsiderable and that of wetland use is udge. Environmental footprint or LCA analysis of SCI compared to classical marine or terrestrial ingredients start to be published.
See the introductive comments on the costs of ingredients
Reference
Check carefully the reference for instance Rosas et al 2019 is missing.
Author Response
Reviewer 2
5/5 - Is the work a significant contribution to the field?
4/5 - Is the work well organized and comprehensively described?
5/5 - Is the work scientifically sound and not misleading?
3/5 - Are there appropriate and adequate references to related and previous work?
5/5 - Is the English used correct and readable?
Q2.1 The review of Glencross is a relatively comprehensive update on the use of single cell ingredients as protein and lipid materials in fish feeding. It is relevant for fish and shrimp nutrition research as there is a growing interest for these ingredients in the context of the development of sustainable aquafeeds. It is well adapted to the scope of Fishes journal.
A2.1 Thanks. ?
Q2.2 The authors considered the main groups of Single cell ingredients and their two main utilisation as protein ingredients and oil ingredients in aquafeed. The review is well adapted to fish feeding and nutrition with different chapters and illustrations related to the main crucial points for including such ingredients in formulation. There is an in-depth analysis of the litterature on utilisation of SCI in the main groups of farmed species salmonids, tilapia, marine species and shrimp. Thus it could be published in Fishes.
A2.2 Again, thanks. ?
Q2.3 There is however a recent review in this field also focused on single cell ingredients in aquaculture Jones et al 2020 Recent advances in single cell protein use as a feed. Current Opinion Biotech. The two reviews are complementary but the authors have to include this review in their analysis, to explain the complementarity and to use as far as possible the information given in this review.
A2.3 We hadn’t seen that review at the time of writing. But have now added reference to it.
Q2.4 The authors did not considered important points for formulation that could be sum up in a specific chapter or in the conclusion : i) the current sources and the costs of these ingredients. It could be useful to have a range of cost for each ingredients compared to fish meal, soya bean meal for SCP and to fish oil and vegetable oil for SCO ii) the pro and the cons of including SCI in aquafeed
A2.4 We deliberately stayed away from the issue of current sources and specific costs as these are quite country and vendor specific things and less so technical or nutritional issues, which was what we were asked to focus on.
Scope of the review :
Q2.5 The classification of single cells organism as microalgae, fungi (yeast) and bacteria does not consider a separate protists group that comprise for instance schizochitrium
A2.5 Schizochytrium was an unusual SCI as appears to have had a variety of classifications over time and doesn yet appear to be fully resolved. The most recent one is what we included in Table 1, where it fits under the Phylum of Heterokonta, which most taxonomists now place as a microalgae. For a summary of that story see: [https://en.wikipedia.org/wiki/Schizochytrium].
Q2.6 It is not clear if the biofloc group includes the mixtures of microalgae produced on CO2 capture from industrial wastes. There are now numerous reviews on these technologies and some of these comprise the characteristics of bioflocs.
A2.6 That may be the case, but in this review, we are focussing on those SCI resources that have had some application/assessment as a feed ingredient in an aquaculture feed. We did not find any biofloc / mixed microalgal studies that fitted our review criteria.
Characterisation of SCI
Q2.7 The characterization is only limited to groups up to species but it has to include known strains varieties or lines and their purity level. For instance selected lines start to be used for Spirulina which is reared since a long term.
A2.7 Agreed, and in other materials (e.g. crop products) this is advocated. However, we are largely limited by the literature here and most never detail the strain details. We will amend to make a comment on this issue.
Q2.8 The potential variability of the characteristics related to rearing conditions of SCI anaerobic or aerobic, bioreactor vs open-field is not considered
A2.8 Noted. But we have mentioned the issue of production system variations as an important part of the characterisation (see lines 108 – 113).
Digestibility
Q2.9 The chapter did not considered the effect of SCI on the microbiote and the contribution of microbiote to the known positive effects of SCI
A2.9 We didn’t include this in the digestibility section as we don’t feel it is pertinent to digestibility, but rather have included it in the immunological/health section (lines 1111-1116), where we feel it is more pertinent.
Q2.10 Digestibility of SCP in Mustela vison can be cited in the text but not could not be included in the table.
A2.10 Agreed.
Processing effect
Q2.11 This is only a simple attempt to review of the effect of SCI processing and on the expected quality of SCI for formulation : availability, requirement, control, management. This chapter could be beneficially enlarged.
A2.11 Yes, but as mentioned there is limited information of the effects of SCI on the production of pellets. This section focuses on examining the feed manufacturing aspects, we only wanted to make small comment on the processing of SCI themselves (lines 1172-1188) as we feel that this area needs a completely new review which is beyond the scope of what we are trying to present.
Health and immunity
Q2.12 The chapter is only focused on positive effects of SCI. There are however negative effects. For instance micro-algae or cyanophycae can synthetised toxins depending on condition of rearing.
A2.12 We didn’t find any aquaculture studies reporting such issues. Guidance?
Q2.13 l279-287 Why not including in this chapter polysaccharides mannan & glucan of yeast that also stimulate immune system. This is only mentionned in the general presentation of each group of SCI.
A2.13 We do mention this in the Immunological and Health Allied Assessments section at lines 1100-1107. In many cases the mannan and b-glucan work is hard to separate out from the yeast biomass work. So there is also a degree of cross-over here with the Utilization and Palatability section.
Next steps The scope of this chapter is limited.
Q2.14 Why not mentionning the need for research on the rôle of microbiote, the effect of specific SCI substrates ex poly-hydroxy-butyrate …
A2.14 This section is not exhaustive. But we have amended to pick up some of these ideas.
Q2.15 This chapter considered only a part of the environmental impact of the production of SCI. The ingredients are produced in various condition from landless pilot plants up to openfield. The environmental impact of plants is not inconsiderable and that of wetland use is udge. Environmental footprint or LCA analysis of SCI compared to classical marine or terrestrial ingredients start to be published.
A2.15 Agree on the pertinence of LCA as an element and as such we had made mention of that in the “Next Steps” section. Probably too early to be able to put in much useful data on this yet.
Q2.16 See the introductive comments on the costs of ingredients
A2.16 See comments about speculating on ingredient costs. Technical versus economic issues…
Reference
Q2.17 Check carefully the reference for instance Rosas et al 2019 is missing
A2.17 Amended.
Reviewer 3 Report
Reviewer comments on the manuscript titled: The application of single cell ingredients in aquaculture feeds – A review
The submitted manuscript is a comprehensive review of the nutrient composition of SCI as well as their results when used in feeding trials with aquatic organisms, focusing on salmonids, tilapia, shrimp and due to less research on marine species in this topic, the marine species have been treated as a separate category. The review appears to present a thorough picture of the status of knowledge on this topic, pointing out areas of special interest for further research in the text. The text is mostly well-written, interesting and easy to follow, although some sections need a bit more work on the wording. The references have some errors, as indicated below. Here are more specific comments:
-Abstract:
Not sure about this sentence structure « something a generalised dichotomy», consider rewriting.
-Line 94: “were among”
-Lines 134-136: So this is of course not always an disadvantage but rather components that could possibly be separated or used together with the rest of the yeast cells in for example functional feeds, like you describe later in this review.
-Lines 206-207: could you revise this sentence” Among the various resources examined, the essential amino acid (EAA) levels reasonably abundant levels of leucine and lysine, though were relatively deficient in histidine (Table 2).”
The sentence after is also very long and could be split up. You also use a lot of words like reasonably, relatively, in this part making the sentences long and with these types of words not being very specific in their meaning (so while something might be a good source of something or not, then by calling something a reasonably good source, leaves it a bit up for interpretation).
-Lines 254-256: would be interesting to see some typical values of EPA and DHA in these sources, and not only the “extreme” value given in the sentence after.
-Line 265: state the goal of this genetic manipulation, just in parenthesis maybe?
-Line 267: “was” produced, I assume?
-Line 466: successfully completely, a bit strange to read, so maybe change to something like “successfully achieve a complete replacement of ”
-Line 480: “that it had”
-Line 518: EPA in muscle? Please specify in the text
-Lines 574-576: I believe this sentence can become clearer if re-written a bit
-Line 586: “and still showed differences” and 80% of what? This is not completely clear, same two lines down. I guess it is the R2 value given here, but this could be clearer in the text. Maybe something more in the line of “x % of the variation in the xx was explained by yy)
-Line 596-599: this sentence would be improved by changing a bit, it doesn’t flow very well. Concerning the following sentence, was the EPA and DHA content higher in the SCO diet than in the FO diet or was just the tissue deposition of these FA more efficient in the SCO diet?
-Lines 613-616: this sentence uses the word “these” four times and could do with a re-writing
-Line 632-633 Inclusion ranged from 20g/kg up to 212g/kg in diets that were either based on a whole or defatted squid meal bases respectively. – so when you write respectively you mean that the 20g/kg diet used a whole squidmeal and the 212 used a defatted squidmeal? The study design is not completely clear to me from the text.
-Line 635-636: rewrite for clarity, three what? “were not significant effects” – That would rather be “had no significant effects”.
-Line 691: Without checking the published data in this topic I would assume that most SCI have high consumer acceptance, so how does this make fungi exceptional? Maybe you could also include something short here on how the listed properties of fungi makes it better/more efficient/more plausible for use in diets than the other SCI discussed in your review?
-717-718: improved compared to the other SCI or to a control feed?
-729-733: You write that over 40% replacement (of fish meal?) reduced performance and then go on to write that using 112 and 180g/kg in diets gave no such effects. You should use the same measures on both so that we can compare, because when written like this we do not know which concentrations (g/kg) a 40% replacement would reach.
-751: genetically modified to contain/produce (whatever you feel fits best)
-759: delete “and in inclusion in animal feeds”? The main point here is that yeast can be produced on waste streams, or am I mistaken?
-766: lower than what?
-770: immunostimulant
-773-780: be clearer in what is being replaced by the yeast and what is higher or improved compared to what. This whole paragraph is a bit unclear, like “However, similar to fish, FCR was improved when shrimp were fed diets with 180 and 250g/kg inclusion.” You write similar to fish, so you mean fish in the diets or are you referring to trials performed on fish (but which fish, marine fish, tilapia, salmon..) and did they use exactly these concentrations and this product? Everything is rather unclear.
-Line 811: re-formulate this part “as an alternative to fishmeal and in combination to”
-834: linear decrease, but you mention only one point of yeast inclusion so between which points is this line drawn? Should be more than two points for any other change than a linear one.
-838: feed formulation is important for all fish species, not just tilapia.
In this same paragraph you are also a bit contradictive, since you first say that in tilapia you must use a very high quality diet as a control and then you say that the positive effects of test ingredients might have been masked by high fish meal?
-970: two times “also” in the same sentence
-988-989: lacking one (
-1014-1015: report what effects were found to be significant when increasing bacterial meal
-1023: what do you mean with “to some extent” here? Maybe you could explain this in the text
-1024: “are highly diverse”
-1051-1052: give the inclusion level used by Kuhn et al.
-1060: “have also been used”
-1067: just write “in both these studies” or something like that.
-1084-1085: inclusion levels?
-1129: “variety of immune system functions”
-Way forwards: In the end you have a very nice summary of the state of potential use of SCI, including some thoughts about economic feasibility as well as thoughts on production methods. You also mention the multiple step method to evaluate ingredients by glencross (2020 paper is not yet available so I am not sure what it says, but assume it is describing the approach you are taking here?) where you mention that only few ingredients (if any) would check off at all points. You do, however, not mention which of these might be the SCI with the highest potential (and then for which species they most likely would have the best use as feed per today). And of the SCI closest to being potentially used in aquaculture, what remains for them to be able to be used? This could give some direction to future research in this field, by having some sort of a common goal. It would be an interesting final of the review paper, as you also mention this step-wise assessment in the start of the paper, thus closing the loop a bit here.
-Table 3: value missing for 18:1n-7 kousolaki 2016. Some zero values given as 0 and others as 0.0, is this a typo or due to level of accuracy reported by the work you are citing?
-Table 4: correct “pasteurised”
Commercial? Have the authors specified anything about the method used at the commercial facilities where they bought it from?
-References:
There are several references that are mentioned in the text that are not found in the list of bibliography, to mention some: belay 1996, rosas 2019, perera 1995, silva neto 2012, macias-sancho 2014.
There are also some references that are written a bit differently, so Øverland is sometimes written in the text like that and sometimes like Overland, would be best to always write it the same way.
There are references missing in the text, but existing in the bibliography (like abdel-tawwab et al.), others are cited wrong in the text (achupallas is written in the text as achupallas et al. 2015, 2016, while in your bibliography both are written to be from 2016; adissin et al. 2019 is written in the text, while it in the reference list is listed as published in 2020; Azim & Little it is written in the text while there are really three authors in this paper).
I would recommend you to have a look at all the references in the text as well as your citation list, as these are just some of the errors I found in the first two pages of the reference list and while randomly selecting some citations to check.
Author Response
Reviewer 3
5/5 - Is the work a significant contribution to the field?
5/5 - Is the work well organized and comprehensively described?
5/5 - Is the work scientifically sound and not misleading?
4/5 - Are there appropriate and adequate references to related and previous work?
4/5 - Is the English used correct and readable?
Q3.1 The submitted manuscript is a comprehensive review of the nutrient composition of SCI as well as their results when used in feeding trials with aquatic organisms, focusing on salmonids, tilapia, shrimp and due to less research on marine species in this topic, the marine species have been treated as a separate category. The review appears to present a thorough picture of the status of knowledge on this topic, pointing out areas of special interest for further research in the text. The text is mostly well-written, interesting and easy to follow, although some sections need a bit more work on the wording. The references have some errors, as indicated below. Here are more specific comments:
A3.1 Thanks. ?
-Abstract:
Q3.2 Not sure about this sentence structure « something a generalised dichotomy», consider rewriting.
A3.2 Amended
Q3.3 -Line 94: “were among”
A3.3 Amended
Q3.4 -Lines 134-136: So this is of course not always an disadvantage but rather components that could possibly be separated or used together with the rest of the yeast cells in for example functional feeds, like you describe later in this review.
A3.4 Noted, but I think we have captured this.
Q3.5 -Lines 206-207: could you revise this sentence” Among the various resources examined, the essential amino acid (EAA) levels reasonably abundant levels of leucine and lysine, though were relatively deficient in histidine (Table 2).”
A3.5 Amended
Q3.6 The sentence after is also very long and could be split up. You also use a lot of words like reasonably, relatively, in this part making the sentences long and with these types of words not being very specific in their meaning (so while something might be a good source of something or not, then by calling something a reasonably good source, leaves it a bit up for interpretation).
A3.6 Amended. But it is sometimes difficult to describe levels of something like EAA other than in relative terms to other resources, hence the use of the term “relatively”.
Q3.7 -Lines 254-256: would be interesting to see some typical values of EPA and DHA in these sources, and not only the “extreme” value given in the sentence after.
A3.7 We couldn’t find any other data other than those references given. So hard to substantiate anything different.
Q3.8 -Line 265: state the goal of this genetic manipulation, just in parenthesis maybe?
A3.8 Amended as suggested.
Q3.9 -Line 267: “was” produced, I assume?
A3.9 Amended
Q3.10 -Line 466: successfully completely, a bit strange to read, so maybe change to something like “successfully achieve a complete replacement of ”
A3.10 Amended
Q3.11 -Line 480: “that it had”
A3.11 Amended
Q3.12 -Line 518: EPA in muscle? Please specify in the text
A3.12 Amended
Q3.13 -Lines 574-576: I believe this sentence can become clearer if re-written a bit
A3.13 Amended
Q3.14 -Line 586: “and still showed differences” and 80% of what? This is not completely clear, same two lines down. I guess it is the R2 value given here, but this could be clearer in the text. Maybe something more in the line of “x % of the variation in the xx was explained by yy)
A3.14 Amended
Q3.15 -Line 596-599: this sentence would be improved by changing a bit, it doesn’t flow very well. Concerning the following sentence, was the EPA and DHA content higher in the SCO diet than in the FO diet or was just the tissue deposition of these FA more efficient in the SCO diet?
A3.15 Amended
Q3.16 -Lines 613-616: this sentence uses the word “these” four times and could do with a re-writing
A3.16 Amended
Q3.17 -Line 632-633 Inclusion ranged from 20g/kg up to 212g/kg in diets that were either based on a whole or defatted squid meal bases respectively. – so when you write respectively you mean that the 20g/kg diet used a whole squidmeal and the 212 used a defatted squidmeal? The study design is not completely clear to me from the text.
A3.17 Amended
Q3.18 -Line 635-636: rewrite for clarity, three what? “were not significant effects” – That would rather be “had no significant effects”.
A3.18 That should have been ‘there’, not “three”. Amended.
Q3.19 -Line 691: Without checking the published data in this topic I would assume that most SCI have high consumer acceptance, so how does this make fungi exceptional? Maybe you could also include something short here on how the listed properties of fungi makes it better/more efficient/more plausible for use in diets than the other SCI discussed in your review?
A3.19 Amended.
Q3.20 -717-718: improved compared to the other SCI or to a control feed?
A3.20 Control diet. Now clarifeid.
Q3.21 -729-733: You write that over 40% replacement (of fish meal?) reduced performance and then go on to write that using 112 and 180g/kg in diets gave no such effects. You should use the same measures on both so that we can compare, because when written like this we do not know which concentrations (g/kg) a 40% replacement would reach.
A3.21 This does in some respect reflect some of the issues seen in the literature, where some scientists are obsessed with fishmeal replacement rather than the effective inclusion of alternatives. There is a distinct difference that many seem to overlook. I’ve tried to clarify this somewhat.
Q3.22 -751: genetically modified to contain/produce (whatever you feel fits best)
A3.22 It already says, “include high levels of EPA and DHA”.
Q3.23 -759: delete “and in inclusion in animal feeds”? The main point here is that yeast can be produced on waste streams, or am I mistaken?
A3.23 Amended.
Q3.24 -766: lower than what?
A3.24 Amended
Q3.25 -770: immunostimulant
A3.25 Amended
Q3.26 -773-780: be clearer in what is being replaced by the yeast and what is higher or improved compared to what. This whole paragraph is a bit unclear, like “However, similar to fish, FCR was improved when shrimp were fed diets with 180 and 250g/kg inclusion.” You write similar to fish, so you mean fish in the diets or are you referring to trials performed on fish (but which fish, marine fish, tilapia, salmon..) and did they use exactly these concentrations and this product? Everything is rather unclear.
A3.26 Amended
Q3.27-Line 811: re-formulate this part “as an alternative to fishmeal and in combination to”
A3.27 Amended
Q3.28 -834: linear decrease, but you mention only one point of yeast inclusion so between which points is this line drawn? Should be more than two points for any other change than a linear one.
A3.28 Amended.
Q3.29 -838: feed formulation is important for all fish species, not just tilapia.
A3.29 Amended
Q3.30 In this same paragraph you are also a bit contradictive, since you first say that in tilapia you must use a very high quality diet as a control and then you say that the positive effects of test ingredients might have been masked by high fish meal?
A3.30 Amended.
Q3.31 -970: two times “also” in the same sentence
A3.31 Amended
Q3.32 -988-989: lacking one (
A3.32 Amended
Q3.33 -1014-1015: report what effects were found to be significant when increasing bacterial meal
A3.33 Amended.
Q3.34 -1023: what do you mean with “to some extent” here? Maybe you could explain this in the text
A3.34 c
Q3.35 -1024: “are highly diverse”
A3.35 Amended
Q3.36 -1051-1052: give the inclusion level used by Kuhn et al.
A3.36 Amended
Q3.37 -1060: “have also been used”
A3.37 Amended
Q3.38 -1067: just write “in both these studies” or something like that.
A3.38 Amended
Q3.39 -1084-1085: inclusion levels?
A3.39 Amended
Q3.40 -1129: “variety of immune system functions”
A3.40 Amended
Q3.41 -Way forwards: In the end you have a very nice summary of the state of potential use of SCI, including some thoughts about economic feasibility as well as thoughts on production methods. You also mention the multiple step method to evaluate ingredients by glencross (2020 paper is not yet available so I am not sure what it says, but assume it is describing the approach you are taking here?) where you mention that only few ingredients (if any) would check off at all points. You do, however, not mention which of these might be the SCI with the highest potential (and then for which species they most likely would have the best use as feed per today). And of the SCI closest to being potentially used in aquaculture, what remains for them to be able to be used? This could give some direction to future research in this field, by having some sort of a common goal. It would be an interesting final of the review paper, as you also mention this step-wise assessment in the start of the paper, thus closing the loop a bit here.
A3.41 Agreed. A good suggestion. We have added a bit to try and cover this now.
Q3.42 -Table 3: value missing for 18:1n-7 kousolaki 2016. Some zero values given as 0 and others as 0.0, is this a typo or due to level of accuracy reported by the work you are citing?
A3.42 The 18:7 level wasn’t reported separately in Kousoulaki et al 2016. The zero values was a formatting issue. Now corrected.
Q3.43 -Table 4: correct “pasteurised”
A3.43 Amended
Q3.44 Commercial? Have the authors specified anything about the method used at the commercial facilities where they bought it from?
Q3.44 In most cases no they didn’t (hence the whole issue about characterisation).
-References:
Q3.45 There are several references that are mentioned in the text that are not found in the list of bibliography, to mention some: belay 1996, rosas 2019, perera 1995, silva neto 2012, macias-sancho 2014.
A3.45 Amended
Q3.46 There are also some references that are written a bit differently, so Øverland is sometimes written in the text like that and sometimes like Overland, would be best to always write it the same way.
A3.46 Amended
Q3.47 There are references missing in the text, but existing in the bibliography (like abdel-tawwab et al.), others are cited wrong in the text (achupallas is written in the text as achupallas et al. 2015, 2016, while in your bibliography both are written to be from 2016; adissin et al. 2019 is written in the text, while it in the reference list is listed as published in 2020; Azim & Little it is written in the text while there are really three authors in this paper).
A3.47 Amended.
Q3.48 I would recommend you to have a look at all the references in the text as well as your citation list, as these are just some of the errors I found in the first two pages of the reference list and while randomly selecting some citations to check.
A3.48 Amended
