A Comparison of Three Protein Sources Used in Medium-Sized Litopenaeus vannamei: Effects on Growth, Immunity, Intestinal Digestive Enzyme Activity, and Microbiota Structure

Round 1

Reviewer 1 Report

This study addressed and interesting area with respect speficially to comparison of three protein sources used in medium-sized Litopenaeus vannamei: Effects of on growth, immunity, intestinal digestive enzyme activity and microbiota structure. This species of shrimp is extensively raised in Asia and Latin America. We have much information on its fundamental nutritional requirements, but more work is needed. The manuscript provides a novel study of merit to examine SCP type protein sources and to examine growth response, immune paprameters and undertake links to gut microbiome. This has been done very well with comprehensive and deep data sets obtained. The science is executed according to what is necessary and is relevent to the experimental trial. It is well explained and in detail. The molecular biology and methods for gut microbiota NGS sequencing and bioinformatic interpretation clearly shows expertise in the area. the use of enzyme assay for specific biomarkers to quantify the interaction of diets on the immune system as well as the direct measurements of key enzymes is paramount to the investigation. Overall this is a robust integration of disciplines to yield added interest to this work. However it would have been useful to make a stronger statement regarding the choice of protein sources within the context of other traditional high protein ingredients used for shrimp feeds. I would also ideally have liked a commercial style diet or a control diet included to the experiment for comparison using mainstream mixed ingredients as in a shrimp diet for vannamei as used in China or Asia. Are Chlorella sorokiniana (CHL) and Clostridium autoethanogenum protein (CAP) as the main protein sources likley to be produced at a sufficent scale in China and elsewhere to justify their choice here? Explain! What is the cost benefit analysis and are these sources realistic in practice for intensive aquaculture production of shrimp requiring a large volume of feed? You could add this to the end of your discussion as a point of interest? Also one thing on line 383 in the discussion, you note 'immune enzymes'. It would be better to say 'immune related enzymes' or 'enzymes associated with the non-specific immune system' as immune enzymes are not really a valid expression.

In general, this is a good study and adds to our knowledge base of the complex association of diet, shrimp immune metabolism, functionality and especially effects on modulation of the gut microbiome and interactions on systemic physiological aspects. Please refine and consider my suggestions for definition of the novel protein ingredient availability and practical use for the industry in aquafeeds.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper ¨Comparison of three protein sources used in medium-sized Litopenaeus vannamei: Effects of on growth, immunity, intestinal digestive enzyme activity and microbiota structure¨ presents novel information regarding the use of new protein sources for the culture of white shrimp. However, you need to clarify some methodological aspects and certain corrections throughout the work.

It must describe in detail the way in which the experimental ingredients have been added (i.e. what were the concentrations of each treatment in relation to the cellular culure, was it used dry, dehydrated, fresh, lyophilized (as was the process of each one), at what time of feed preparation process was added, how it was added (cover, during mixing, when leaving the extruder), etc. Describe the process of each treatment in different paragraphs.

Why in the paragraph of the methodology there is a concentration of FM and in the table it is different (if it is in percentage it is equal to grams per kilo). Why is there the difference?

Separate in different paragraphs the experimental conditions and the culture conditions.

In addition, it should be clarified and added:

-How often the water changes were made, it is not clear what they mean by 1/3 of a day (every 8 hours)?

-photoperiod

-feed rate? (in relation to biomass)

 Throughout the text, you must review and correct the way in which you are citing, since in many cases (i.e. L161), the way in which commas and points are placed is not appropriate (review the way to cite correctly). Make all the necessary changes throughout the work.

A general review is recommended throughout the text.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf