Effect of Calanus finmarchicus Hydrolysate Inclusion on Diet Attractiveness for Whiteleg Shrimp (Litopenaeus vannamei)

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Thank you for your interest and for investing your time in the manuscript. We are sure your questions and suggestions will improve the quality of the final article. Please find our replies below:

1. It is a very good suggestion to emphasize the research question and the significance. We’ve added the following part to the last paragraph of the introduction:

The primary objective of this research is to rigorously evaluate the potential chemoattractive effects of C. finmarchicus hydrolysate as a supplement in shrimp feed formulations. The study aims to quantify the impact of this novel zooplankton-derived ingredient on the feeding behavior and intake levels of whiteleg shrimp. The ultimate goal is to identify an effective strategy to enhance the palatability of plant protein-rich diets and thereby optimize the growth performance and sustainability of shrimp aquaculture practices.

2. Feed intake was calculated by the mass difference between distributed and uneaten feed, which was collected and weighed after each feeding time. FCR was calculated (1.76) as a collective parameter for all diets in the trial (Table S2 in supplementary data), individual values for each diet were not possible due to the daily tank rotation study design (to exclude any tank bias). The feed amount distributed each time was adjusted according to stocking density, mean body weight, and the intervals between feeding times (can be found in chapter 2.3. Feeding trial).

The manuscript has now been updated to include all of this information.

3. Agreed. The following paragraph now replaces the methodology (lines 156-164) with more general findings in the study:

The whiteleg shrimp fully accepted all the diets from the start (mean body weight 3.67 g) and throughout the experiment until the end (mean body weight 10.46 g). The daily rotation study design (Figure 1) of switching the diets to new tanks each day was also successful. This specific design excluded any potential tank bias in the study, but also meant that only collective performance parameters could be measured beyond feed intake. All the parameters were well within acceptable ranges; survival in the experiment was 99.6 %, specific growth rate was 4.18 %/day, and the feed conversion ratio was 1.76. The rest of the collective performance parameters can be found in Table S2 of the supplementary data.

4. We argue in the discussion that the water-soluble nature, the low-molecular weight peptides, and the levels of chemoattractive amino acids (taurine, glycine) are important in explaining why calanus hydrolysate has the ability to increase the attractiveness of shrimp diets.

5. This is possible if required. It was omitted to avoid making the figure legends too long (e.g. describing Kruskal-Wallis vs. ANOVA), and all of the statistical analyses are described in chapter 2.4. Statistics.

6. Table and figure legends have now been updated with n = replicates, wherever appropriate.

7. Agreed and corrected.

Reviewer 2 Report

The manuscript Effect of Calanus finmarchicus hydrolysate inclusion on diet attractiveness for whiteleg shrimp (Litopenaeus vannamei) investigates the potential chemoattractive effect of a zooplankton hydrolysate on a shrimp diet, by analysing its increased feed intakes. This study is well organized and provides insightful and practical information on additives that can be used by feed producers to enhance the palatability of plant-based shrimp feeds. The experimental design and the methodology employed are appropriate for the goal of this study and I have only a few remarks that could help improve this manuscript.

Why is there a “water” row in table 2? I have never seen a formulation table with the amount of water and it seems a bit odd, especially since most of the values are negative.

Also, regarding the formulation table, there is no details on the specific composition of individual ingredients used in the feeds, this should be included in the table footnotes.

In line 116, the word “till” is used, instead of “until”, which is incorrect.

Author Response

Dear reviewer,

Thank you for your interest and for investing your time in the manuscript. We are sure your questions and suggestions will improve the quality of the final article.

The “water” row has been removed. It was a remnant from the formulation process, thank you for correcting this.

All the information that is available on the individual ingredients have now been added to the footnotes of the formulation table.

“Till” has been replaced by “until” in line 116.

Reviewer 3 Report

Although unequal variance found in the low FM group, I see the trial desigh with 3 main factors: FM level CH level and KH level. A 3 way anova shold be performed. Perhaps the interaction between FM and hydrolisate addtion would be discovered and would support the fact that hydrolisate is not working in low FM but it is in high FM incluison

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Thank you for your interest and for investing your time in the manuscript. We are sure your questions and suggestions will improve the quality of the final article.

You bring up an excellent point. We actually did try a two-way ANOVA, using low/high FM as one factor and ingredient inclusion rate as the second factor. It did not reveal any interactions, probably due to the unequal variances of the low FM group.

While we acknowledge the potential for discovering interactions between FM level and the inclusions, we determined that pursuing a non-parametric equivalent of a two or three-way ANOVA may not be appropriate due to the lack of normality assumption in a substantial portion of our dataset. Considering these limitations, we believe we have chosen the most suitable analytical approach to present our findings. Specifically, we have applied the non-parametric Kruskal-Wallis test for the low FM group and an ordinary one-way ANOVA with Tukey post-hoc analysis for the high FM group. We believe that these methods provide robust insights into the effects of ingredient inclusion rate across different FM levels, while accounting for the variability inherent in our data.