Round 1

Reviewer 1 Report

Animals 2141198

The nature of the study described in the manuscript is appropriate for the journal; the way the authors designed their study, decided on the data to collect, and presented the material makes the manuscript eligible for publication.

The way the authors presented their material is satisfactory, as is the structure of the manuscript and the way the authors presented their data. 

The title indicates the content of the manuscript.

Keywords are appropriate.

The presentation of the Abstract is precise and complete. It includes brief details on study objectives, experimental design, sampling protocols and data analysis, and some quantitative information to allow readers to see the size of treatment effects.

The introduction is clear and concise and contains sufficient background information for readers to understand why the study was deemed necessary. The Introduction closes with a clear statement of the objectives of the study.

The description of the M&M is complete. The presentation of the M&Ms is clear to see exactly how the authors have done the job. Information provided on study design and culture conditions is sufficient, and information provided on sampling and testing routines is accurate. The amount of information provided on M&Ms is sufficient for readers to see exactly how the study was designed, how sampling was performed, what protocols were used, how calculations were performed, and how the data was analyzed.

The discussion is supported by the experimental data.

Some of the points that require attention:

Lines 162-164: Why was a ‘restricted’ feeding regime adopted, instead of feeding to satiation at each meal?

Lines 164-165: Report the mean value of the water temperature 

Lines 165-166: How was uneaten feed collected? Manually or using an appropriate device?

Lines 224-225: Can you describe the anatomical part? anterior gut tract and posterior gut tract 

Line 284-289: Table 3 -conform to table 2, adding the measurement units

Line 294-295: Table 4 - conform to table 2, adding the comma after each parameter described, before the measurement units

Line 335-336: Table 5 – conform to table 2. Some commas are missing; remove the brackets

The study did not include a digestibility trial, so there is no information on how treatments affected the bioavailability of feed nutrients. However, it would be interesting if the authors reported proximal chemical composition analysis of both groups of fish to evaluate the effects of treatment on nutrient retention and utilization efficiency.

Author Response

Animals 2141198-R1

Dear Reviewer,

We would like to thank for your revisions of our paper ID: Animals 2141198 “Influence of Hermetia illucens larvae meal dietary inclusion on growth performance, gut histological traits and stress parameters in Sparus aurata” for your valuable comments and advice that helped us to improve our manuscript.

All of your comments were duly considered and all of them were accepted. Revisions were included in the manuscript and highlighted in red. Below, we provide our point-by-point response to you.

Our answers are signed in red.

 

Reviewer 1

Animals 2141198

The nature of the study described in the manuscript is appropriate for the journal; the way the authors designed their study, decided on the data to collect, and presented the material makes the manuscript eligible for publication.

The way the authors presented their material is satisfactory, as is the structure of the manuscript and the way the authors presented their data. 

The title indicates the content of the manuscript.

Keywords are appropriate.

The presentation of the Abstract is precise and complete. It includes brief details on study objectives, experimental design, sampling protocols and data analysis, and some quantitative information to allow readers to see the size of treatment effects.

The introduction is clear and concise and contains sufficient background information for readers to understand why the study was deemed necessary. The Introduction closes with a clear statement of the objectives of the study.

The description of the M&M is complete. The presentation of the M&Ms is clear to see exactly how the authors have done the job. Information provided on study design and culture conditions is sufficient, and information provided on sampling and testing routines is accurate. The amount of information provided on M&Ms is sufficient for readers to see exactly how the study was designed, how sampling was performed, what protocols were used, how calculations were performed, and how the data was analyzed.

The discussion is supported by the experimental data.

Some of the points that require attention:

Lines 162-164: Why was a ‘restricted’ feeding regime adopted, instead of feeding to satiation at each meal?

 

The aim of this study's experimental design is to reproduce more possible real conditions of an aquaculture farming to obtain results immediately transferable at commercial level. In the industrial aquaculture farming, food is administered "at ration" so to limit wastefulness and consequent increasing of rearing costs.

Below an example of feeding scheme used for gilthead seabream (Gruppo Veronesi).

 

 

Below, some recent researches in which feed was administered at ration.

Pulido et al. 2022. Effect of dietary black soldier fly larvae meal on fatty acid composition of lipids and sn-2 position of triglycerides of marketable size gilthead sea bream fillets. Aquaculture.

Caimi et al. 2022. Dietary inclusion of a partially defatted black soldier fly (Hermetia illucens) larva meal in low fishmeal-based diets for rainbow trout (Oncorhynchus mykiss). Journal of Animal Science and Biotechnology.

Chemello et al. 2020. Partially Defatted Tenebrio molitor Larva Meal in Diets for Grow-Out Rainbow Trout, Oncorhynchus mykiss (Walbaum): Effects on Growth Performance, Diet Digestibility and Metabolic Responses. Animals.

 

Lines 164-165: Report the mean value of the water temperature

The values of water oxygen, pH and temperature were reported (lines 169-170).

 

Lines 165-166: How was uneaten feed collected? Manually or using an appropriate device?

Feed was collected by a syphon. Some details were added (lines 182-183).

 

Lines 224-225: Can you describe the anatomical part? anterior gut tract and posterior gut tract

DONE (lines 238-240)

 

Line 284-289: Table 3 -conform to table 2, adding the measurement units DONE

 

Line 294-295: Table 4 - conform to table 2, adding the comma after each parameter described, before the measurement units DONE

 

Line 335-336: Table 5 – conform to table 2. Some commas are missing; remove the brackets DONE

 

The study did not include a digestibility trial, so there is no information on how treatments affected the bioavailability of feed nutrients. However, it would be interesting if the authors reported proximal chemical composition analysis of both groups of fish to evaluate the effects of treatment on nutrient retention and utilization efficiency. DONE (lines 382-385)

 

Kind regards,

Marianna Oteri

Corresponding Author

Researcher of Animal Nutrition

University of Messina

Department of Veterinary Sciences, Messina - IT

Author Response File: Author Response.docx

Reviewer 2 Report

The submitted work on the effect of partial fishmeal substitution with BSF meal in Sparus aurata is interesting. So many work has been reported in line to this in other species. In fact, the evaluation of substitute such as BSF can harness global ingredient sustainability to reduce impacts on fishmeal harvest. The experimental design and data analysis is performed to conclude a strong recommendation. Still, the manuscript need a major re-organization and changes as highlighted to clear the readers understanding. My comments are appended below.

1. In summary, Line 33-34: No need to expand in abstract, simply write n=?...performed in triplicates. Rewrite the sentence. 

2. Line 36:  gut histological tract...change this to gut histology

The abstract section need serious rewriting. Be concise and present the key findings only.

Introduction

1. Line 62. Please cite reference for this statement. 

2. Line 81-82: Reference needed.

3. Your work focus much on gut morphology and stress. Few examples of earlier research in this line need to be incorporated in review. 

4. Whether work has been done on Sparus? If this is the first report, mention that here.

5. No mention on the economic importance of the fish experimented is mentioned in introduction. Highlight why you have chosen the species.

Methods

1. Line 161: define the photoperiod frame here.

2 Line 165. How was the uneaten feed removed. Was it segregated from the fecal output?

3. Line 176, 190: No need to mention twice how many fishes were sampled. Always write the n values.

4. Line 210: Was 2500 g centrifugation enough to separate the plasma? Seems less. If suitable methods are available, cite the reference.

5. What was the magnification for microscopic examination of gut? Define that.

Data analysis. Mention the data presentation way. Was data normalised?

Results

1. Table 4 & 6. If data were analysed, better indicate significant values as superscripts.

2. In all micrographs, indicate the magnification levels.

Discussion.

1. Line 406. Focus on plasma parameters.

2. No need to expand Hermetia illucens meal in repeated places. 

Conclusion.

You did not highlight the growth. Our major focus is growth...mention this here.

Conclusion is weak. Please rewrite with a strong conclusion.

 

 

 

 

 

 

 

 

Author Response

Animals 2141198-R1

Dear Reviewer,

We would like to thank for your revisions of our paper ID: Animals 2141198 “Influence of Hermetia illucens larvae meal dietary inclusion on growth performance, gut histological traits and stress parameters in Sparus aurata” for your valuable comments and advice that helped us to improve our manuscript.

All of your comments were duly considered and all of them were accepted. Revisions were included in the manuscript and highlighted in red. Below, we provide our point-by-point response to you.

Our answers are signed in red.

 

Reviewer 2

The submitted work on the effect of partial fishmeal substitution with BSF meal in Sparus aurata is interesting. So many work has been reported in line to this in other species. In fact, the evaluation of substitute such as BSF can harness global ingredient sustainability to reduce impacts on fishmeal harvest. The experimental design and data analysis is performed to conclude a strong recommendation. Still, the manuscript need a major re-organization and changes as highlighted to clear the readers understanding. My comments are appended below.

1. In summary, Line 33-34: No need to expand in abstract, simply write n=?...performed in triplicates. Rewrite the sentence. The abstract section was rewritten (lines 30-43).
2. Line 36:  gut histological tract...change this to gut histology DONE (line 32)

The abstract section need serious rewriting. Be concise and present the key findings only. DONE (lines 30-43)

Introduction

1. Line 62. Please cite reference for this statement. DONE (line 64)
2. Line 81-82: Reference needed. DONE (line 83)
3. Your work focus much on gut morphology and stress. Few examples of earlier research in this line need to be incorporated in review. DONE (lines 93-95)
4. Whether work has been done on Sparus? If this is the first report, mention that here. DONE (lines 103-106)
5. No mention on the economic importance of the fish experimented is mentioned in introduction. Highlight why you have chosen the species. DONE (lines 96-102)

Methods

1. Line 161: define the photoperiod frame here.

Feeding trial lasted from February to August and photoperiod, in this time, has naturally changed. We have added details on months and latitude at which feeding trial was carried out. (lines 177-178)

2 Line 165. How was the uneaten feed removed. Was it segregated from the fecal output?

Feed was collected by syphoning 15 minutes after each meal, so it was not mixed with fecal output. Some details were added (lines 182-183).

3. Line 176, 190: No need to mention twice how many fishes were sampled. Always write the n values. DONE (lines 193, 206, 219)
4. Line 210: Was 2500 g centrifugation enough to separate the plasma? Seems less. If suitable methods are available, cite the reference.

2500 × g for 15 minutes was enough speed to separate plasma from whole blood. To obtain plasma the  indications of protocol by Cusabio (Fish cortisol ELISA kit) were followed. Below, some papers in which blood was centrifuged at the same speed to obtain plasma (lines 224-225).

1. Carbajal A. et al. 2019. Comparative assessment of cortisol in plasma, skin mucus and scales as a measure of the hypothalamic-pituitary-interrenal axis activity in fish. Aquaculture;
2. Samaras at al. 2021. Cortisol concentration in scales is a valid indicator for the assessment of chronic stress in European sea bass, Dicentrarchus labrax L. Aquaculture;
3. Oyarzun R. at al. 2020. Stocking density affects the growth performance, intermediary metabolism, osmoregulation, and response to stress in Patagonian blennie Eleginops maclovinus – Aquaculture.
4. Vargas-Chacoff L. et al. 2016. Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch) display differential metabolic changes in response to infestation by the ectoparasite Caligus rogercresseyi – Aquaculture
5. What was the magnification for microscopic examination of gut? Define that. DONE (at line 247-248)

Data analysis. Mention the data presentation way. Was data normalised?

The sentence was reformulated for better explained the presented results. All data were checked for normal distribution and homogeneity of variances and normalized. (Lines 265-266)

Results

1. Table 4 & 6. If data were analysed, better indicate significant values as superscripts.

All data were analysed. In table 4 no significant differences were found either for the diet (D) or for the final body weight (FBW) and there are no superscripts. In tables 3, 5 and 6 the significant values have been indicated as superscripts. Thank you for your suggest.

2. In all micrographs, indicate the magnification levels. DONE (line 338)

Discussion.

1. Line 406. Focus on plasma parameters. DONE (line 426)
2. No need to expand Hermetia illucens meal in repeated places. DONE

Conclusion.

You did not highlight the growth. Our major focus is growth...mention this here. DONE (lines 516-517, 521-522)

Conclusion is weak. Please rewrite with a strong conclusion. The conclusion section was rewritten (Lines 513-526).

Kind regards,

Marianna Oteri

Corresponding Author

Researcher of Animal Nutrition

University of Messina

Department of Veterinary Sciences, Messina - IT

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have carefully reconstructed the content as per suggestions. My further comment that need to be taken care are

1. Line 103-106: they report that few works have been done in similar line. A line should follow this on how your work fills the gap. Was any research gap observed that led to this work? 

Line 170: O2 should be written as DO

Line 248: why did you use different magnification, while in results section you indicated only one (10x)? 

Rest are fine. 

 

Author Response

Animals 2141198-R2

Dear Reviewer,

We would like to thank for your revisions of our paper ID: Animals 2141198 “Influence of Hermetia illucens larvae meal dietary inclusion on growth performance, gut histological traits and stress parameters in Sparus aurata” for your valuable comments and advice that helped us to improve our manuscript.

All of your comments were duly considered and all of them were accepted. Revisions were included in the manuscript and highlighted in red. Below, we provide our point-by-point response to you.

Our answers are signed in red.

Reviewer 2

The authors have carefully reconstructed the content as per suggestions. My further comment that need to be taken care are

Line 103-106: they report that few works have been done in similar line. A line should follow this on how your work fills the gap. Was any research gap observed that led to this work? Updated studies using HIM in Sparus aurata diets reported results in fish growth and nutrient utilization, but nothing on gut morphology or stress parameters (Karapanagiotidis et al., 2014). Other studies have evaluated only the qualitative characteristics of Sparus aurata fillets fed with different levels of HI inclusion (Pulido et al., 2022). In this study, which is part of a research project, we evaluated the suitability of incorporating various levels of HI by evaluating growth performance parameters, gut histology and stress parameters (as reported in the aim of the study, lines 107-112), as well as the chemical and microbiological composition of fillets, as reported by Oteri et al. (2022), previously.

 

Furthermore, also the levels of HIM inclusion were different:

Karapanagiotidis et al., 2014:                     HIM10: 95 g/kg, HIM20: 194 g/kg, HIM30: 276 g/kg

Pulido et al. 2022:                                         HIM9: 92 g/kg; HIM18: 184 g/kg; HIM27: 276 g/kg

In our study:                                                  HIM25: 79 g/kg, HIM35: 110 g/kg, HIM50: 157 g/kg

Line 170: O2 should be written as DO. DONE

Line 248: why did you use different magnification, while in results section you indicated only one (10x)? For histological examinations, the possible changes between groups were evaluated under different magnifications (5x, 10x and 40x). However, the best images of the alterations of transverse section of anterior intestine of S. aurata were obtained using a magnification of 10x.

Kind regards,

Marianna Oteri

Corresponding Author

Researcher of Animal Nutrition

University of Messina

Department of Veterinary Sciences, Messina - IT

Author Response File: Author Response.docx