Inulin Supplementation in Diets for Tropical Gar (Atractosteus tropicus) Larvae: Effects on Growth, Survival, and Digestive and Antioxidant Enzyme Activities
, Jenny F. López-Hernández 1, Otilio Méndez-Marín 1, Susana C. De la Rosa-García 1, Emyr S. Peña-Marín 2, Dariel Tovar-Ramírez 3, Cesar A. Sepúlveda-Quiroz 1, Graciela M. Pérez-Jiménez 1, Luis D. Jiménez-Martínez 4, Gloria G. Asencio-Alcudia 1,* and Carlos A. Álvarez-González 1,*Round 1
Reviewer 1 Report
The remarks are indicated here and highlight in the pdf file.
Review: Inulin Supplementation in Diets for Tropical Gar (Atractosteus 2 tropicus) Larvae: Effects on Growth, Survival, Digestive and 3 Antioxidant Enzyme Activities.
The comments on this MS are given below. However, in the MS, they are highlighted on the corresponding lines.
In general,
MS must rewrite in several sections and several inconsistencies must be explained.
Abstract.
Line 26-28: Clarify the feeding protocol.
Line 38: the antioxidant activity was higher with control diet (see lines 27-28), not with inulin supplementation.
Introduction.
Focus it more deeply on the use of inulin in fish feeding.
Line 48: it is something wrong.
Line 65-66: rewrite this sentence.
Materials and Methods.
Line 78: Check it.
Line 80-84: instead, state that accession numbers of datasets will be provided during review process or prior publication.
Line 86: larviculture.
Line 103-105: The feeding protocol is not clear enough.
Line 111: in line 99, authors refer to microparticulate food, but in this section, they don’t mention how the microparticulate food was prepared (they obtain 5 mm pellets). And it is not clear how the inulin was incorporated to the feed.
Dry ingredients had the same particle diameter?
Line 129-130: the SGR formula is incorrect. It is: (lnWf - lnWi x 100) / t. The SGR data must be recalculated.
Line 143: check it.
Line 147: Indicate if the measurement were done by duplicate, triplicate!
Line 148: if at the end of trial six larvae were sampled for experimental unit, why only three larvae digestive system were used per experimental unit?
Line 151 and 158: it is recommended to use ‘’g’’ instead of rpm.
Line 185: … to analyze survival, …
Line 187-188: it is a bit confusing this sentence. Rewrite this section (2.9).
Results.
Why for weight and length use medians ± RI and for survival, enzyme and antioxidant activities mean ± SD?
Line 195-197: however final weight for diet 0 was not significant different for that of diets 0.5, 2.0 and 2.5 (according to figure), bien only different with diets 1.0 and 1.5. This paragraph should be rewrite accordingly. Define the term RI.
Line 202-203: data on weight gain and SGR are not given.
Line 204: where it is defined ‘r percentage’? Just used %.
Line 210-212: this is not completely true, they are not different from diet 1.0 (amylase) and 1.5 (lipase), respectively.
Line 219: correct title of vertical axis (survival).
Line 225: Fig. 3. The units in the title are wrong.
Line 235. Fig. 4. Why units’ enzymes are different from those expressed in lines 177-180?
Discussion.
The authors state that inulin inclusion improved, in general, digestion and absorption, production of short-chain fatty acids, and several vitamins; but they don’t explain why the growth from inulin treatments (weight gain, SGR) was not different from that of control (that were even a bit higher).
It must focus on the study results, showing evidence that support them. Not explaining the positive correlations between inulin inclusion with growth and oxidative enzymes that are opposite to the findings in this research.
Authors did not explain why, 1) catalase activity is lower with inulin inclusion than control? Why superoxide dismutase activity decreased with inulin inclusion as compared to control? Why glutathione peroxidase activity tended to be higher only at 2.5% inulin?
Line 240-242: this is not completely true. There are some diets with lower inulin (than 2.0) that are not significant different from the mentioned ones (see figure 1). Rewrite this paragraph accordingly.
Line 258-259: how the levels used in these studies compare with those used for you?
Line 272: it is the colon differentiated in the intestine of a fish? Please refers to it or explain your reasoning better.
Line 286: ‘’were’’
Line: 296-302: too long paragraph, being difficult to read and understand. Make it in shorter sentences.
Line 308-309: this is a repeated idea (similar at line 293-294).
Line 322-325: this is not true. Your results indicated the contrary: an increase in oxidative enzymes at lower inclusion level of inulin (o zero inclusion).
Line 314-341: in this paragraph, authors cited references supporting the fact that inulin inclusion increases the antioxidant enzymes activity, however, they found the contrary. In this context, they must focus to explain their results, why inulin inclusion reduced the antioxidant activity? This a filling paragraph.
Line 325-331: A long paragraph! Try to rewrite in short sentences for better reading.
Line 342-347: Again, another paragraph supporting the beneficial effect of dietary inulin inclusion, facts that are not in agreement with author findings. Another filling paragraph.
Line 349-350: this was not measured by authors (active substances by microbiome) to state here.
Line 358-365: another long paragraph, that make it difficult to understand. Try to make short sentences.
Conclusions
Line: 369-370: This is not completely true. Despite there is an increase in CAT activity with inulin inclusion, it is not higher than control, so there is not a real improvement or beneficial effect of inulin.
Author Response
We thank for your comments and suggestions to improve our manuscript. Changes from reviewer 1 are highlighted in green.
The comments on this MS are given below. However, in the MS, they are highlighted on the corresponding lines.
In general,
MS must rewrite in several sections and several inconsistencies must be explained.
Response: We appreciate the reviewer's suggestion, and the article was re-revised by the authors.
Abstract.
Line 26-28: Clarify the feeding protocol.
Response: Done
Line 38: the antioxidant activity was higher with control diet (see lines 27-28), not with inulin supplementation.
Response: Corrected
Introduction.
Focus it more deeply on the use of inulin in fish feeding.
Response: Thanks for the comment, we have adjusted this section according to your suggestion.
Line 48: it is something wrong.
Response: We have adjusted the paragraph.
Line 65-66: rewrite this sentence.
Response: We have rewritten the sentence
Materials and Methods.
Line 78: Check it.
Response: It is correct since the beginning, it’s the protocol we use in Mexico, and has been published in previous papers.
Line 80-84: instead, state that accession numbers of datasets will be provided during review process or prior publication.
Response: Edition mistake, we deleted this paragraph already.
Line 86: larviculture.
Response: Corrected
Line 103-105: The feeding protocol is not clear enough.
Response: The protocol has been adjusted.
Line 111: in line 99, authors refer to microparticulate food, but in this section, they don’t mention how the microparticulate food was prepared (they obtain 5 mm pellets). And it is not clear how the inulin was incorporated to the feed.
Response: We added the missing information in line 119-120.
Dry ingredients had the same particle diameter?
Response: Yes, we added the information.
Line 129-130: the SGR formula is incorrect. It is: (lnWf - lnWi x 100) / t. The SGR data must be recalculated.
Response: Sorry for the misunderstood, we corrected the formula in line 129, one of the parentheses were missed.
Line 143: check it.
Response: Already checked.
Line 147: Indicate if the measurement were done by duplicate, triplicate!
Response: We clarify the measurement in line 152.
Line 148: if at the end of trial six larvae were sampled for experimental unit, why only three larvae digestive system were used per experimental unit?
Response: We added the information in line 134, we use three individuals for antioxidant analysis and three for digestive analysis.
Line 151 and 158: it is recommended to use ‘’g’’ instead of rpm.
Response: Thank for your recommendation, we have converted and switched.
Line 185: … to analyze survival, ….
Response: Switched.
Line 187-188: it is a bit confusing this sentence. Rewrite this section (2.9).
Response: We have adjusted the section prior to be more precise.
Results.
Why for weight and length use medians ± RI and for survival, enzyme and antioxidant activities mean ± SD?
Response: We preferred to use this representation because the data (length, weight and GPx) doesn’t comply with the postulates of parametric statistics and the rest of the data, does it.
Line 195-197: however final weight for diet 0 was not significant different for that of diets 0.5, 2.0 and 2.5 (according to figure), bien only different with diets 1.0 and 1.5. This paragraph should be rewrite accordingly. Define the term RI.
Response: The term of RI it described in the text (line 198), meaning of the median value.
Line 202-203: data on weight gain and SGR are not given.
Response: Corrected, we deleted line 128.
Line 204: where it is defined ‘r percentage’? Just used %.
Response: We switched the word to value.
Line 210-212: this is not completely true, they are not different from diet 1.0 (amylase) and 1.5 (lipase), respectively.
Response: In those lines, we said that the treatments who shows differences are 2 y 2.5%, we sorry for the misunderstood.
Line 219: correct title of vertical axis (survival).
Response: Done
Line 225: Fig. 3. The units in the title are wrong.
Response: Sorry for the mistake, but the units are correct, because we normalized the data with Bradford technique.
Line 235. Fig. 4. Why units’ enzymes are different from those expressed in lines 177-180?
Response: Because all the antioxidant enzymes were normalized with Bradford technique.
Discussion.
The authors state that inulin inclusion improved, in general, digestion and absorption, production of short-chain fatty acids, and several vitamins; but they don’t explain why the growth from inulin treatments (weight gain, SGR) was not different from that of control (that were even a bit higher).
It must focus on the study results, showing evidence that support them. Not explaining the positive correlations between inulin inclusion with growth and oxidative enzymes that are opposite to the findings in this research.
Authors did not explain why, 1) catalase activity is lower with inulin inclusion than control? Why superoxide dismutase activity decreased with inulin inclusion as compared to control? Why glutathione peroxidase activity tended to be higher only at 2.5% inulin?
Response: We are totally agreed with the comments of the reviewer and we have focused on the clear evidence that support the positive effects in A. tropicus larvae fed inulin. Thanks.
Line 240-242: this is not completely true. There are some diets with lower inulin (than 2.0) that are not significant different from the mentioned ones (see figure 1). Rewrite this paragraph accordingly.
Response: We have rewritten this paragraph.
Line 258-259: how the levels used in these studies compare with those used for you?
Response: We found reports suggesting that 2 and 2.5% of supplementation provides higher survival rates, that those with lowest concentration of inulin.
Line 272: it is the colon differentiated in the intestine of a fish? Please refers to it or explain your reasoning better.
Response: We change the word colon to posterior intestine.
Line 286: ‘’were’’.
Response: Corrected
Line: 296-302: too long paragraph, being difficult to read and understand. Make it in shorter sentences.
Response: We divided the paragraph to make it shorter.
Line 308-309: this is a repeated idea (similar at line 293-294).
Response: We short the sentence to emphasize the importance of inulin in the digestion process that we found in this study.
Line 322-325: this is not true. Your results indicated the contrary: an increase in oxidative enzymes at lower inclusion level of inulin (o zero inclusion).
Response: We have added a citation accordingly with our statement.
Line 314-341: in this paragraph, authors cited references supporting the fact that inulin inclusion increases the antioxidant enzymes activity, however, they found the contrary. In this context, they must focus to explain their results, why inulin inclusion reduced the antioxidant activity? This a filling paragraph.
Response: We have corrected and added the missing citation.
Line 325-331: A long paragraph! Try to rewrite in short sentences for better reading.
Response: Thank you for noticing, we have divided in short sentences.
Line 342-347: Again, another paragraph supporting the beneficial effect of dietary inulin inclusion, facts that are not in agreement with author findings. Another filling paragraph.
Response: Thank you for your observation, we consider needed this paragraph in order to show the differences found in our study vs previous studies with different species of fish.
Line 349-350: this was not measured by authors (active substances by microbiome) to state here.
Response: Although we have not measured it, there is evidence of the effect of inulin directly on the microbiome, which we can observe through the improved digestive capacity that was analyzed.
Line 358-365: another long paragraph, that make it difficult to understand. Try to make short sentences.
Response: We have fragmented the paragraph.
Conclusions
Line: 369-370: This is not completely true. Despite there is an increase in CAT activity with inulin inclusion, it is not higher than control, so there is not a real improvement or beneficial effect of inulin.
Response: We have corrected this paragraph.
Reviewer 2 Report
The article presents a study on the supplementation of inulin in the diet of tropical gar larvae. It deals with the effects on growth, survival, digestion and the very important activities of antioxidant enzymes. The study is interesting and will provide valuable information for tropical gar breeding programmes, especially for breeding larvae during their development and for when dietary changes should be made. I have carefully reviewed the paper and would say that it is excellent and suitable for publication. From this point of view this paper contains a lot of new scientific data. The paper presents an original study about incorporation of of inulin into the larvae of A. tropicus, which increases survival and improves the activities of acid, alkaline, amylase, and lipase proteases. I recommend that the paper can be published with minor corrections.
The title and abstract are representative, clear and concise. The title describes the study very well. The abstract succinctly includes the main points of the study. The keywords (inulin; tropical gar ) are included in the title, so other keywords should be chosen.
The introduction is well written, clear and concise. The purpose of the study is clearly defined.
Line 111th - the dot must be deleted after the title
The experiments are well designed and conducted with appropriate statistical analyses. The SI international system of units of measurement is used correctly. The quality of the illustrations is good. The design of the study is appropriate. The results are clear and informative and are presented clearly. The results are credible. The discussion is relevant and complete. The conclusions are based on the results and clearly summarise the major findings and contributions of the study.
The manuscript contains sufficient and appropriate references. References are cited according to the guidelines. The reference list corresponds to the references in the text and vice versa.
Author Response
We thank for your comments and suggestions to improve our manuscript. Changes from reviewer 2 are highlighted in blue.
The article presents a study on the supplementation of inulin in the diet of tropical gar larvae. It deals with the effects on growth, survival, digestion and the very important activities of antioxidant enzymes. The study is interesting and will provide valuable information for tropical gar breeding programs, especially for breeding larvae during their development and for when dietary changes should be made. I have carefully reviewed the paper and would say that it is excellent and suitable for publication. From this point of view this paper contains a lot of new scientific data. The paper presents an original study about incorporation of of inulin into the larvae of A. tropicus, which increases survival and improves the activities of acid, alkaline, amylase, and lipase proteases. I recommend that the paper can be published with minor corrections.
The title and abstract are representative, clear and concise. The title describes the study very well. The abstract succinctly includes the main points of the study. The keywords (inulin; tropical gar) are included in the title, so other keywords should be chosen.
Response: Thanks for your comment.
The introduction is well written, clear and concise. The purpose of the study is clearly defined.
Response: Thanks for your comment.
Line 111th - the dot must be deleted after the title.
Response: Done
The experiments are well designed and conducted with appropriate statistical analyses. The SI international system of units of measurement is used correctly. The quality of the illustrations is good. The design of the study is appropriate. The results are clear and informative and are presented clearly. The results are credible. The discussion is relevant and complete. The conclusions are based on the results and clearly summarize the major findings and contributions of the study.
The manuscript contains sufficient and appropriate references. References are cited according to the guidelines. The reference list corresponds to the references in the text and vice versa.
Response: Thanks for your comment.
Reviewer 3 Report
This is an interesting study that investigated how Inulin supplementation in diets for tropical gar larvae and its effects on growth, survival, digestive and antioxidant enzyme activities. I have few major suggestions that the authors may wish to consider:
1. Break the introduction into 2 or 3 paragraphs
2. Please check the statement “Fish nutrition.” This can be deleted or rephrase better
3. Please delete lines 80 to 84 from the manuscript not needed
4. In figure 4C there is no statistical sign any reason for not adding the letters as done in other figures?
Author Response
We thank for your comments and suggestions to improve our manuscript. Changes from reviewer 3 are highlighted in yellow.
This is an interesting study that investigated how Inulin supplementation in diets for tropical gar larvae and its effects on growth, survival, digestive and antioxidant enzyme activities. I have few major suggestions that the authors may wish to consider:
- Break the introduction into 2 or 3 paragraphs.
Response: Thank you for your advice, we have done it.
- Please check the statement “Fish nutrition.”
Response: This can be deleted or rephrase better
- Please delete lines 80 to 84 from the manuscript not needed.
Response: We consider keeping the information because it’s important for us to let the readers know where the larvae are coming from our breeding fish.
- In figure 4C there is no statistical sign any reason for not adding the letters as done in other figures?
Response: We haven't added the letters because no significant differences were found in GPx antioxidant enzymatic activity.
Reviewer 4 Report
This work addresses the importance of adding prebiotics such as inulin in farmed fish diets, using as model, tropical gar, a local species with economical relevance in Mexico. The work has been well conducted and the authors present interesting results that can be quite helpful in the context of improving health/welfare of this species. I suggest that the manuscript can be published after reviewing the aspects indicated below.
Introduction
Lines 46-48. These sentences need further reviewing by the authors, as they are not clear and/or incomplete e.g., “That is way methods have been sought to counteract this damage (what type of methods? Physiological-, molecular-, phenotype-based?, or simply meaning the use of prebiotics and probiotics?
“Fish nutrition (4). “ ???
Line 62, do you mean “improved” growth instead of “improve”
Lines 65-66 “which has allowed it to close (????) or “disclose” its cultivation cycle?
Lines 68-70. The whole sentence needs further reviewing as it sounds confusing and/or incomplete.
Materials and Methods
Lines 80-84. This paragraph regarding instructions for authors should not be here in the manuscript.
Line 190, ”which is why they” is redundant, please remove it.
Results
Lines 198- 201. Please reformulate this sentence in a more objective way, reporting to differences that are statistically significant or not, for total length, when comparing diets. “Statistically equal” is not acceptable. The differences/or similarities in total lengths were (or not) statistically different….In fact, there are differences or not, and there are similarities or not for a given parameter under analysis, that are or not significant…
Lines 209-210, again, differences are higher or lower and you may report to p-values in () for indicating the level of significance of the difference
Lines 230-231, again, “highest values (p < 0.05); please refer to values that were significantly different when comparing control with the diets with higher inclusion levels of inulin…
Lines 260-264. The paragraph starts with “Which” and repeats again “Which” in the rest of the sentence. The whole sentence is too large and should be reviewed.
Conclusions
Indeed, it was observed a tendency to increased levels of CAT activity with increasing levels of inulin in the diets, but even so, lower (and statistically different) in respect to the control. On the other hand, SOD activity decreased and not increased at higher concentrations of inulin, being significantly lower compared with the control. So, it is not totally clear the relationship between inulin levels and antioxidant capacity at the tested levels, only that seems to exist a tendency to an increase of CAT at higher concentrations of inulin (increasing from 0.5-2.5%). Therefore, the authors should be more cautious in this conclusion, and review it.
Author Response
We thank for your comments and suggestions to improve our manuscript. Changes from reviewer 4 are highlighted in pink.
This work addresses the importance of adding prebiotics such as inulin in farmed fish diets, using as model, tropical gar, a local species with economical relevance in Mexico. The work has been well conducted and the authors present interesting results that can be quite helpful in the context of improving health/welfare of this species. I suggest that the manuscript can be published after reviewing the aspects indicated below.
Response: Thanks for your comments.
Introduction
Lines 46-48. These sentences need further reviewing by the authors, as they are not clear and/or incomplete e.g., “That is way methods have been sought to counteract this damage (what type of methods? Physiological-, molecular-, phenotype-based?, or simply meaning the use of prebiotics and probiotics?.
Response: We have added and corrected the sentence.
“Fish nutrition (4). “ ???
Response: We corrected this section.
Line 62, do you mean “improved” growth instead of “improve”.
Response: Thank you for noticing, we have corrected.
Lines 65-66 “which has allowed it to close (????) or “disclose” its cultivation cycle?
Response: This term refers to the fact that thanks to the investigations carried out by the work team it has allowed us to close the biological cycle of cultivation (reproduction, hatchery, nursery, and grow-out).
Lines 68-70. The whole sentence needs further reviewing as it sounds confusing and/or incomplete.
Response: We have replaced the sentence, thank you for noticing.
Materials and Methods
Lines 80-84. This paragraph regarding instructions for authors should not be here in the manuscript.
Response: Thank you for your observation, we have eliminated this paragraph.
Line 190, ”which is why they” is redundant, please remove it.
Response: Thank you for your contribution, we have removed it.
Results
Lines 198- 201. Please reformulate this sentence in a more objective way, reporting to differences that are statistically significant or not, for total length, when comparing diets. “Statistically equal” is not acceptable. The differences/or similarities in total lengths were (or not) statistically different…. In fact, there are differences or not, and there are similarities or not for a given parameter under analysis, that are or not significant…
Response: We have reformulated and added the missing words to avoid confusions.
Lines 209-210, again, differences are higher or lower and you may report to p-values in () for indicating the level of significance of the difference.
Response: We have mentioned.
Lines 230-231, again, “highest values (p < 0.05); please refer to values that were significantly different when comparing control with the diets with higher inclusion levels of inulin…
Response: Thanks for your observation, however we mentioned (line 209-215) that for acid and alkaline proteases, larvae fed with the inclusion of 2.5% of inulin shows the highest activities and for amylase and lipase the highest activity was for treatments 2 and 2.5% of inulin supplementation.
Lines 260-264. The paragraph starts with “Which” and repeats again “Which” in the rest of the sentence. The whole sentence is too large and should be reviewed.
Response: We reviewed the paragraph and split it
Conclusions
Indeed, it was observed a tendency to increased levels of CAT activity with increasing levels of inulin in the diets, but even so, lower (and statistically different) in respect to the control. On the other hand, SOD activity decreased and not increased at higher concentrations of inulin, being significantly lower compared with the control. So, it is not totally clear the relationship between inulin levels and antioxidant capacity at the tested levels, only that seems to exist a tendency to an increase of CAT at higher concentrations of inulin (increasing from 0.5-2.5%). Therefore, the authors should be more cautious in this conclusion, and review it.
Response: We have corrected the conclusions section, thank you for noticing.
Round 2
Reviewer 1 Report
Dear authors. The comments on your MS are cited below.
The new comments on this MS are given below. These small changes and recommendations to improve the MS.
Abstract.
Line 26-27: the feeding protocol is not clear enough. From 3 to 10 DAH larvae were only fed with nauplii, but the cofeeding period is not clear (it was from 8 DAH to 11 DAH? It must be, with nauplii: from 3 to 7 DAH, cofeeding: from 8 to 11 DAH and balanced diet: from 12 to 21 DAH. I tis correct?
Introduction.
It is ok.
Materials and Methods.
Line 95-96: What was the rate (or proportion) of nauplii and diet during the cofeeding, days 8-11?
Line 182-183: Check it! The specific growth rate was deleted from measurements in section 2.4.
Line 184-185: This sentence it is still a bit confusing.
Results.
Line 192-193: In the way this sentence is written it induces that weight with control diet was significant different from that of diet with 2.5 and 2.0% inulin, fact that is not true (it was similar). The weight from control diet was only different from the other treatments (1.0 and 1.5%).
Line 195-196: it is still confusing. Write control diet instead ´´diet control´´
Line 199: data on weight gain are not showed in the MS. They must be included.
Line 207-208: Amylase activity at 1.0% inulin is lower but not different from that at 2 and 2.5%. Lipase activity is similar at 1, 1.5 and 2% inulin and also between 1.5, 2 and 2.5%, respectively.
Line 223: Fig. 3. The units in the title of figure or in the figure are wrong. They must be (U/mg protein-1).
Line 233. Fig. 4. Why units to express enzyme activity are different from those expressed in M&M for the same enzymes (lines 174-180: CAT (nmol min-1 mL-1), SOD (U mL-1), GPX (nmol min-1 mL-1))? They should be the same in both parts of MS.
Discussion.
The authors state that inulin inclusion improved, in general, digestion and absorption, production of short-chain fatty acids, and several vitamins; but they don’t explain why the growth from inulin treatments (weight gain, SGR) was not different from that of control (that were even a bit higher).
It must focus on the study results, showing evidence that support them. Not explaining the positive correlations between inulin inclusion with growth and oxidative enzymes that are opposite to the findings in this research.
Authors did not explain why, 1) catalase activity is lower with inulin inclusion than control? Why superoxide dismutase activity decreased with inulin inclusion as compared to control? Why glutathione peroxidase activity tended to be higher only at 2.5% inulin?
Line 261: may you indicate the dose of probiotic used those authors (42 and 43).
Line 285: change ´´was´´ by ´were´´.
Line 285-288: in these three lines uses ´´which´´ three times. Try to use other alternative word.
Line: 302-308: Make it in shorter sentences.
Line 324: Perhaps it is better split the sentences after treatment. Start with: It is due to …
Line 329: inulin. In order to …
Line 333: Write: Likewise in lower case or use ¨dot¨.
Author Response
All the reviews from this Reviewer are mark in green in the Ms.
Line 26-27: the feeding protocol is not clear enough. From 3 to 10 DAH larvae were only fed with nauplii, but the cofeeding period is not clear (it was from 8 DAH to 11 DAH? It must be, with nauplii: from 3 to 7 DAH, cofeeding: from 8 to 11 DAH and balanced diet: from 12 to 21 DAH. I tis correct?
RESPONSE: Indeed, its correct. Thank you for your observation, we have corrected.
Introduction.
It is ok.
Materials and Methods.
Line 95-96: What was the rate (or proportion) of nauplii and diet during the cofeeding, days 8-11?
RESPONSE: We started (8 DAH) with 20 nauplii per larvae and ended (11 DAH) with 50 nauplii per day.
Line 182-183: Check it! The specific growth rate was deleted from measurements in section 2.4.
RESPONSE: Checked
Line 184-185: This sentence it is still a bit confusing.
RESPONSE: We have clarified the sentence.
Results.
Line 192-193: In the way this sentence is written it induces that weight with control diet was significant different from that of diet with 2.5 and 2.0% inulin, fact that is not true (it was similar). The weight from control diet was only different from the other treatments (1.0 and 1.5%)
RESPONSE: Thank you so much for your observation, we have changed the sentence and corrected the missing data.
Line 195-196: it is still confusing. Write control diet instead ´´diet control´´
RESPONSE: Done
Line 199: data on weight gain are not showed in the MS. They must be included.
RESPONSE: We have added the missing data.
Line 207-208: Amylase activity at 1.0% inulin is lower but not different from that at 2 and 2.5%. Lipase activity is similar at 1, 1.5 and 2% inulin and also between 1.5, 2 and 2.5%, respectively.
RESPONSE: We have corrected the drafting.
Line 223: Fig. 3. The units in the title of figure or in the figure are wrong. They must be (U/mg protein-1).
RESPONSE: Thank you for the observation. We have corrected.
Line 233. Fig. 4. Why units to express enzyme activity are different from those expressed in M&M for the same enzymes (lines 174-180: CAT (nmol min-1 mL-1), SOD (U mL-1), GPX (nmol min-1 mL-1))? They should be the same in both parts of MS.
RESPONSE: Because in the M&M we described the formula used to obtain activities from the commercial brand used, but we have normalized with Bradford technique, that is the one shows in the graph.
Discussion.
The authors state that inulin inclusion improved, in general, digestion and absorption, production of short-chain fatty acids, and several vitamins; but they don’t explain why the growth from inulin treatments (weight gain, SGR) was not different from that of control (that were even a bit higher).
It must focus on the study results, showing evidence that support them. Not explaining the positive correlations between inulin inclusion with growth and oxidative enzymes that are opposite to the findings in this research.
Authors did not explain why, 1) catalase activity is lower with inulin inclusion than control? Why superoxide dismutase activity decreased with inulin inclusion as compared to control? Why glutathione peroxidase activity tended to be higher only at 2.5% inulin?
Line 261: may you indicate the dose of probiotic used those authors (42 and 43).
RESPONSE: We have indicated. Author 42: 1% and 2% of inulin supplementation. Author 43: 1, 2 and 3% of inulin supplementation (prebiotic)
Line 285: change ´´was´´ by ´were´´.
RESPONSE: Done
Line 285-288: in these three lines uses ´´which´´ three times. Try to use other alternative word.
RESPONSE: Done
Line: 302-308: Make it in shorter sentences.
RESPONSE: Done
Line 324: Perhaps it is better split the sentences after treatment. Start with: It is due to …
RESPONSE: Done, thank you for the advice.
Line 329: inulin. In order to …
RESPONSE: Done
Line 333: Write: Likewise in lower case or use ¨dot¨.
RESPONSE: Done
Reviewer 3 Report
The authors have addressed all my concerns.
Author Response
We appreciate all your comments, hoping they all have been resolved.
We mark in green in the Ms the changes.
