Hemp Meal (Cannabis sativa) as an Alternative Dietary Protein Source for European Perch (Perca fluviatilis)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

L225-227 "Please provide the catalog number of the commercial kit to facilitate the repetition of the experiment."

 

L241 For all descriptions of experimental results, it is recommended to include P values when significant differences exist, to facilitate readers' understanding of the experimental findings.

 

L258 Table 3

Please retain the same number of decimal places for both the mean values and standard errors in the data. Superscript letters should be placed after the standard error values. And, please modify all tables in the manuscript.

 

All data should be presented as mean ± standard error. Please modify the parameters for Rearing indices.

 

L290-294 To facilitate observation of these parameters, please label these parameters in Figure 1.

 

L304 Is the gene expression result presented as relative expression level? Relative expression level requires using one treatment group as the control, with its expression set to 1. However, according to the results shown in Figure 2, none of the treatment groups has an expression value of 1. Why is this? Please verify your experimental data.

 

L317 Figure 2D not figure 2.

 

L341 It is best to clearly indicate which is the male parent and which is the female parent for the hybrid.

 

L347-350 Could these contradictory results be related to whether the hemp meal was defatted or not? Please verify whether the hemp meal used in each study had undergone defatting processing.

 

L359-360 First, please clarify whether the hemp meal used in the current experiment was defatted, and provide the proximate composition data of the hemp meal, including crude protein, crude fat, etc. Second, although hemp oil, hemp residues, and hemp panicle extract all originate from hemp, they are essentially three completely different feed ingredients. Providing such examples is therefore inappropriate.

 

 

Author Response

L225-227 "Please provide the catalog number of the commercial kit to facilitate the repetition of the experiment."
We added the missing kit numbers, as requested.

L241 For all descriptions of experimental results, it is recommended to include P values when significant differences exist, to facilitate readers' understanding of the experimental findings.
We added the exact p-values (up to 4 digits) throughout the Results, whenever there was a significant difference mentioned.

L258 Table 3
Please retain the same number of decimal places for both the mean values and standard errors in the data. Superscript letters should be placed after the standard error values. And, please modify all tables in the manuscript.
We corrected the decimals and superscripts throughout all Tables, as requested.

All data should be presented as mean ± standard error. Please modify the parameters for Rearing indices.
We added the ±SEM values for all rearing indices, as requested.

L290-294 To facilitate observation of these parameters, please label these parameters in Figure 1.
The studied parameters are not uncommon in histological research of the digestive system and we refer to past papers in the M&Ms, in which they were visually explained. Hence, we decided not to follow this suggestion as it would affect the clarity of this image panel. We hope for your understanding.

L304 Is the gene expression result presented as relative expression level? Relative expression level requires using one treatment group as the control, with its expression set to 1. However, according to the results shown in Figure 2, none of the treatment groups has an expression value of 1. Why is this? Please verify your experimental data.
In response to the first question: no, the data was not presented in relation to the 0% control group, but only as expression levels normalized to the housekeeping genes, hence the different values even for the control. However, after further consideration we decided to change these graphs to indeed show the relative expression values, i.e. the 0% group now always has the value of 1. We also introduced changes in Figure 2 footnote to accomodate this new feature.

L317 Figure 2D not figure 2.
We decided to introduce similar precise lettering for both Figures throughout sections 3.3, 3.4 and 3.5.

L341 It is best to clearly indicate which is the male parent and which is the female parent for the hybrid.
The Authors of the study in question did not provide such information in that paper.

L347-350 Could these contradictory results be related to whether the hemp meal was defatted or not? Please verify whether the hemp meal used in each study had undergone defatting processing.
We are very grateful for this suggestion. Unfortunately, none of the three papers which we commented upon specified whether the HM was defattened, or not. Nevertheless, we added a sentence mentioning this issue after the sentence in question, at the end of the paragraph.

L359-360 First, please clarify whether the hemp meal used in the current experiment was defatted, and provide the proximate composition data of the hemp meal, including crude protein, crude fat, etc. Second, although hemp oil, hemp residues, and hemp panicle extract all originate from hemp, they are essentially three completely different feed ingredients. Providing such examples is therefore inappropriate.
Yes, we did use defatted hemp protein powder. We added this information at the beginning of section 2.1, along with the basic proximate composition of this hemp meal and other ingredients in the new Table 1. Regarding the second issue, we deleted that entire sentence, as suggested.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript addresses an interesting and timely topic regarding the replacement of fish meal with plant-based meals in European perch. Although no significant effects were observed on the primary outcome of growth performance, the study provides valuable insights into other physiological and biochemical parameters and may serve as useful preliminary data for future research. The following comments are offered to help improve the overall quality and clarity of the manuscript.

1. Typographical and minor errors

• Line 176: Please correct “idenfication” to “Identification.”
• Table 2: Please revise the table title from “RT-PCR” to “RT-qPCR.”
• Line 351: The term “blood plasma” should be revised to ensure appropriate terminology.

2. Simple Summary

• Line 42: The potential reduction in feed costs resulting from replacing fish meal with hemp meal is an important practical consideration and may be briefly addressed in the Discussion. In this context, issues related to hemp meal quality control and any potential additional costs should also be considered.

3. Methodology

• Please clearly specify which part(s) of the hemp plant were used in the diet formulation.
• Based on Line 160, if 10 fish per group were sampled, the following corrections are required:
• Line 186: Revise “8 sampled individuals per group (n = 160)” to “10 sampled individuals per group (n = 200).”
• Line 188: Revise “8 individuals per group (n = 800)” to “10 individuals per group (n = 1000).”
• Line 193: Revise “(n = 80 for each)” to “(n = 100 for each).”
• Statistical analysis
• The manuscript indicates the use of the Kruskal–Wallis test for several parameters; however, the post hoc test applied following significant results is not specified. Please clarify which multiple-comparison procedure was used.
• For histological parameters, One-Way ANOVA followed by Fisher’s post hoc test is stated. Please confirm that this approach was applied consistently and that the assumptions for parametric analysis were satisfied.

4. Results

• Lines 253–256: Based on Table 3, differences in fat, protein, and ash contents among treatments appear minimal and largely overlapping; therefore, statements referring to “highest” values may not be statistically justified.
• Table 3 (Line 259): Please move “Results are presented as means ± SEM” to the table footnote.
• Table 4 (Line 268): Please move “Results are presented as means ± SD” to the table footnote.
• Table 4: Consider using standard abbreviations for triglycerides and cholesterol (e.g., TG and TC).
• Table 5 (Line 289): Please move “Results are presented as means ± SD” to the table footnote.
• Line 297: The statement regarding PFH, PEH, and PSH does not appear to be consistent with the data in Table 5; the group with the highest values should be HM10 rather than HM20.
• Figures 2, Sections 3.4 (Gene expression analysis) and 3.5 (Analysis of hepatic enzymes): The figures and corresponding text suggest that comparison strategies other than standard post hoc pairwise tests (e.g., grouped or hierarchical comparisons) may have been applied; however, this is not clearly described in the Methods section. Moreover, the practical relevance of grouping treatment levels for statistical comparison is unclear, as it does not evidently enhance biological or applied interpretation. Clarifying the statistical approach and its rationale, or alternatively presenting direct group-to-group comparisons, would improve transparency and interpretability.

Author Response

1. Typographical and minor errors

• Line 176: Please correct “idenfication” to “Identification.”
• Table 2: Please revise the table title from “RT-PCR” to “RT-qPCR.”
• Line 351: The term “blood plasma” should be revised to ensure appropriate terminology.
  We corrected these mistakes.
  
  

2. Simple Summary

• Line 42: The potential reduction in feed costs resulting from replacing fish meal with hemp meal is an important practical consideration and may be briefly addressed in the Discussion. In this context, issues related to hemp meal quality control and any potential additional costs should also be considered.
  Thank you for this remark. We improved the text as suggested by adding some insight on the economics of using hemp meal for aquafeed, which we placed at the very end of the Conclusions.
  
  

3. Methodology

• Please clearly specify which part(s) of the hemp plant were used in the diet formulation.
  There is a whole new sentence at the start of section 2.1 which specifies the details regarding the HM used, also including its proximate composition.
  
  
• Based on Line 160, if 10 fish per group were sampled, the following corrections are required:
  • Line 186: Revise “8 sampled individuals per group (n = 160)” to “10 sampled individuals per group (n = 200).”
  • Line 188: Revise “8 individuals per group (n = 800)” to “10 individuals per group (n = 1000).”
  • Line 193: Revise “(n = 80 for each)” to “(n = 100 for each).”
    Thank you for spotting these errors - we corrected them for the proper n=10.
    
    
• Statistical analysis
  • The manuscript indicates the use of the Kruskal–Wallis test for several parameters; however, the post hoc test applied following significant results is not specified. Please clarify which multiple-comparison procedure was used.
    We added the information that we used Dunn's test for multiple comparisons.
    
    
  • For histological parameters, One-Way ANOVA followed by Fisher’s post hoc test is stated. Please confirm that this approach was applied consistently and that the assumptions for parametric analysis were satisfied.
    As it was already stated in the opening sentence in this section (3.7): "The obtained datasets were subjected to normality (Shapiro-Wilk) and homogeneity (Levene) tests". Hence, if a parametric analysis was chosen for some data instead of Kruskal-Wallis, this means that the necessary criteria were indeed met.
    
    

4. Results

• Lines 253–256: Based on Table 3, differences in fat, protein, and ash contents among treatments appear minimal and largely overlapping; therefore, statements referring to “highest” values may not be statistically justified.
  In response, we opted to remove these sentences entirely.
  
  
• Table 3 (Line 259): Please move “Results are presented as means ± SEM” to the table footnote.
• Table 4 (Line 268): Please move “Results are presented as means ± SD” to the table footnote.
• Table 4: Consider using standard abbreviations for triglycerides and cholesterol (e.g., TG and TC).
• Table 5 (Line 289): Please move “Results are presented as means ± SD” to the table footnote.
  We fixed these issues, as requested.
  
  
• Line 297: The statement regarding PFH, PEH, and PSH does not appear to be consistent with the data in Table 5; the group with the highest values should be HM10 rather than HM20.
  Thank you for spotting this crucial typo. Indeed, HM10 showed the highest values of these parameters. We fixed this now.
  
  
• Figures 2, Sections 3.4 (Gene expression analysis) and 3.5 (Analysis of hepatic enzymes): The figures and corresponding text suggest that comparison strategies other than standard post hoc pairwise tests (e.g., grouped or hierarchical comparisons) may have been applied; however, this is not clearly described in the Methods section. Moreover, the practical relevance of grouping treatment levels for statistical comparison is unclear, as it does not evidently enhance biological or applied interpretation. Clarifying the statistical approach and its rationale, or alternatively presenting direct group-to-group comparisons, would improve transparency and interpretability.
  We apologize for this misleading design of our graphs. There was no grouping of data - the brackets were only supposed to reduce the number of lines and stars since we applied the group-to-group Dunn's test which in some cases yielded multiple differences, e.g. pparaa in the anterior intestine differed between groups: HM0 and HM20, HM0 and HM30, but also HM10 and HM20, as well as HM10 and HM30. However, since we had to rework these graphs anyway according to Reviewer 1 (to make the data relative to group HM0), we decided to simply switch to letter superscripts, as it is in Tables. This modification resolves the issue completely. Furthermore, as mentioned before, we enhanced the description of statistical analysis, so there will be no future misunderstandings.

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, the authors investigated the possibility of using hemp meal (HM) as an alternative source of protein in extruded feed for European perch (Perca fluviatilis). Fish were fed with diets containing different levels of HM (0, 10, 20 or 30%, respectively) for 10 weeks. This ms is well-written but some comments should be addressed before its acceptance. The use of two replicates per each treatment is not acceptable and the authors should use three replicates at least. The authors replaced HM with other protein sources; that results in a changeable difference in their amounts in diets. So, the proximate chemical composition of used ingredients should be done and added in a separate table. Also, the amino acids profile of the tested diets should be done and compared with fish requirements of amino acids.

Table 1: The protein contents of Hemp meal, fish meal, Soybean PC, Wheat gluten, Blood meal should be added under Table 1. There are big differences in carbohydrates and fibers contents among diets; that may affect diets digestion.

 Table 3: Add feed intake and calculate FCR instead of FCE to this table. Separate Muscle proximate composition to be in a separate table.

Figure 1: Split this figure to represent each organ separately.

Table 5: Split this table to represent intestine data and liver data in separated tables.

Figure 2: Split this figure to 4 figures to represent each type of data separately.

Check the references list and make sure that all references info is complete and the scientific names are in italic style. For example, the reference [10] is MSc thesis, add the university, and country.

Author Response

The use of two replicates per each treatment is not acceptable and the authors should use three replicates at least.
Dear Reviewer, the use of technical tank replicates for study groups can sometimes be a difficult limitation, as it was in the case of our study. Nevertheless, there are published studies which also involved the use of tank duplicates instead of triplicates, what shows that this is not as big of an issue as labeled by some. This includes our own study which we previously published in this very same journal (10.3390/ANI11123520). We hope for your understanding and move on from this issue as we will not repeat the experiment at this point, due to numerous obvious reasons.

The authors replaced HM with other protein sources; that results in a changeable difference in their amounts in diets. So, the proximate chemical composition of used ingredients should be done and added in a separate table. Also, the amino acids profile of the tested diets should be done and compared with fish requirements of amino acids.
An amino acid profile of diets would indeed by a beneficial addition to this manuscript, but unfortunately it was not made back when we conducted this experiment. Unfortunately, we currently do not have any remaining samples of the diets anymore, and the funding has already expired, which is why we are unable to perform this analysis at this point. As with our response above, we hope for your understanding that we are unable to improve this part of the study now. Meanwhile, some minor differences of ingredients other than HM and FM resulted due to the necessity to achieve full energetic balance of the diets, as well as proper physical properties of the pellets. For clarity, we have created a new Table 1 which compiles the basic proximate composition of the ingredients, as requested.

Table 1: The protein contents of Hemp meal, fish meal, Soybean PC, Wheat gluten, Blood meal should be added under Table 1. There are big differences in carbohydrates and fibers contents among diets; that may affect diets digestion.
Since we created the new Table 1 containing detailed information about the ingredients, we opted not to repeat this information in Table 2 containing feed formulation and composition.

Table 3: Add feed intake and calculate FCR instead of FCE to this table. Separate Muscle proximate composition to be in a separate table.
As requested, we added feed intake and replaced FER with FCR. We also separated muscle contents from the other basic parameters by transferring them to a new Table 5.

Figure 1: Split this figure to represent each organ separately.
Dear Reviewer, we respectfully decided not to follow this suggestion. Given the extensive scope of analyses and the number of measured parameters, this histological panel was intentionally organized in an integrated manner to maintain readability and to avoid excessive fragmentation of visual information. Dividing the figure by organs would reduce clarity, potentially hindering rather than facilitating the interpretation process. We sincerely hope for your understanding of our decision.

Table 5: Split this table to represent intestine data and liver data in separated tables.
As requested, we splitted the data into two tables - one containing intestinal, the other hepatic parameters.

Figure 2: Split this figure to 4 figures to represent each type of data separately.
Similarly to what was mentioned above, this suggestion would result in an unnecessary multiplication of images, which we wanted to avoid. We already reworked the gene expression graphs according to valid suggestions of both Reviewers 1 and 2, but none of these suggestions mentioned anything about creating separate figures for each graph. Hence, we decided to keep all four graphs together, as everything is visible within them, especially now after the change to letter superscripts indicating statistical significance (rather than bars with asterisks).

Check the references list and make sure that all references info is complete and the scientific names are in italic style. For example, the reference [10] is MSc thesis, add the university, and country.
The references were automatically generated using Mendeley. However, we double-checked all citations in our database and corrected all the missing information, as requested, as well as the final formatting in the manuscript file.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No.

Author Response

Since the Reviewer did not write any further suggestions, we believe that the (x) "must be improved" markings pointing towards the results and figures are merely remnants from the first round of review and the Reviewer simply forgot to change them. Hence, we are sincerely grateful that the Reviewer seemingly accepted all of our revisions.

Reviewer 2 Report

Comments and Suggestions for Authors

You may consider further minor revisions as follows:

1. Please carefully check for consistency in writing style throughout the manuscript. For example, ensure consistent use of uppercase and lowercase letters, particularly in the table footnotes.

2. Table 1: In general, details regarding the source or supplier of materials are not typically provided in the table footnotes. Instead, this information would be more appropriately described under the Materials section.

Author Response

1. Please carefully check for consistency in writing style throughout the manuscript. For example, ensure consistent use of uppercase and lowercase letters, particularly in the table footnotes.
   We double-checked the manuscript for these details, as suggested. All letters should always be uppercase.

2. Table 1: In general, details regarding the source or supplier of materials are not typically provided in the table footnotes. Instead, this information would be more appropriately described under the Materials section.
   We thank the Reviewer for this suggestion. In response, we decided to rework this table by adding two new columns "Product" and "Producer/Supplier", and moved the questioned footnotes into these columns. We believe this solution to be the most adequate.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors respond to comments partially, but it is ok. This ms needs the amino acids profile of the tested diets to ensure it satisfies the amino acids requirements of European perch. The figures are not splited to different Figures as recommended previously.

 

Author Response

The authors respond to comments partially, but it is ok. This ms needs the amino acids profile of the tested diets to ensure it satisfies the amino acids requirements of European perch.
As mentioned in our previous round of revision, we are unable to perform this analysis at this point in time, as we:

1. do not have the experimental diets anymore,
2. do not have the funding anymore (the project has been completed and was reported to the funding authorities).

We acknowledge the usefulness of this analysis, but by making this request the Reviewer essentially discredits all the other analyses and animal sacrifices that were made to complete this study. Furthermore, we performed this study in the form and shape which was accepted by the funding authority, as well as the Local Ethical Committee, and nobody had raised this issue before. Hence, we politely ask the Reviewer to accept the situation as it is.

The figures are not splited to different Figures as recommended previously.
Dear Reviewer, as you have thyself mentioned, the splitting of Figures was merely a "recommendation", which is why we respectfully declined some of these changes, although we did follow through with the splitting of tables. In order to find middle ground on this issue we have now decided to split Figure 2 into two, one containing intestinal gene expression, the other containing hepatic gene expression and enzymatic activity. Meanwhile, Figure 1 will remain as it is, since that is the layout that we have chosen originally and we want to stick with it. Lastly, we have now splitted Table 4 into two, one containing fish body parameters, the other gathering rearing indices. We sincerely hope that we have now shown enough good will towards the Reviewer, while also maintaining some of our own intentions regarding this manuscript.