The Effects of Porphyra yezoensis Polysaccharides on Intestinal Health of Spotted Sea Bass, Lateolabrax maculatus

Round 1

Reviewer 1 Report

This manuscript deals with the effect of a polysaccharide from the algae Porphyra. Introduction was well written, focused in the subject of study, but  authors could add a few lines describing the importance of spotted sea bass. Methodology was well described, but some details arte missing in statistical analysis. Results are not properly described (see comments below) and must be rewriten, so I could not evaluate if discussion is correct.  

abstract: remove "(P<0.05)" and "(P>0.05) "

keywords: authors should not use words that are in the title

lines 43-44: I do not think this sentence regarding oxidized fish oil is relevant to introduction

line 73: ...can also reduce and improve...

line 77: add scientific name of spotted sea bass

table 1: which flour was this? Wheat flour?

line 101: replace "was" by "were"

item 2.2: the experiment lasted 52 days. Why authors did not evaluate fish growth? 

lines 119-120: I suppose the intestines that were separated for histological analysis were not frozen in liquid nitrogen, as it seems from this sentence

lines 123-124: all abbreviations must be defined in the first mention

line 130: replace "r/min" by "g"

line 166: it is better not to cite fig. 3 here. The first figure to be cited must be fig. 1

lines 171-173: authors have to indicate how they checked normality and homocedasticity of variances. The test is Duncan's. Have authors tried to check if there is any relationship between PP doses and responses?

lines 178-182: this description is not correct. Fish fed 9g/kg presented higher amylase activity than fish fed control diet. That's all.

lines 183-184: it looks like authors do not believe in their statistical analysis

lines 186-188: delete from ", and the activity..."

lines 189-190: correct, but no relevant here

lines 190-191: too generic. Does starch has a significant role for spotted sea bass feeding?

line 196: Is it the same kind of PP? What is the PP dietary level that increases digestive enzymes activity in grass carp?

lines 203-204: measuring the activity of digestive enzymes does not allow to speculate about intestinal health 

tables 2-5: all abbreviations must be defined

I see no reason to define groups as K, PP1-PP5 - why not just place the PP doses?

correct in the tables: data values with different superscript letters in the same column were significantly different  

I started reading the results of antioxidant capacity and saw that authors followed the same pattern as or digestive enzymes, i.e., they are not following strictingly the results of statistical analysis. Authors must rewrite all results.

English: the text can be understood without too much trouble, but several sentences are not well structured (for example item 2.4), so an English review by a native speaker is suggested

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The use of natural-based feed additives in aquaculture industry has been made from medicinal herbs, and fortunately, today algae (macro, micro, seaweeds) and their derivatives are developing as valuable and unique natural resources. The topic is interesting, innovative, and highly recommended to publish in Fishes. However, there are some comments that need to be clarified by the authors as follows:

Please clarify the species of the rhodophyt seaweeds in the title.

L21: Please notice which genus/species/phylum were the most abundant. The readers are eager to know the composition of the intestinal microbes.

Overall, the English language of the manuscript is native and easy to read and understand. However, please do not forget to use “the” when it is necessary. For instance, in the abstract section, correct to: …the intestinal amylase activity… the intestinal trypsin activity…. in the lipase activity ….. the intestinal glutathione content…. the malondialdehyde content…. the catalase activity…. the intestinal villus morphology…. etc.

L47: Please correct … [9,10], et al.

L61: why the authors mentioned the enzyme hydrolysates from Porphyra which is far from their topic and research purpose.

L63-66: Since there are good literature on the positive dietary effects of seaweed and seaweed polysaccharides in fish, especially the aquaculture species, change the statement for fish.

L69: What is the share of other components?

L71-74: Please withdraw the literature on mammals and terrestrial animals.

Critical comment: The description that how PP was manufactured was missed in the M&M section. It is your main part of your research and it is missed!

Critical comment: How the authors were ensured that they obtained a pure PP? It is your main part of your research and it is missed!

L83: Based on which criteria the doses were selected?

What is zinc oxide eugenol cement liquid? did you used clove oil?

L105: what was the environmental condition? Tm, CO, No2, No3, Ammonia, pH, photoperiod

What was the adaptation period for the fish to the new site?

Is any specific reason to rear the fish up to 52 days? In most of the studies it is common to culture the fish at least 56 days (8 weeks)

L123-127: All the acronyms should be explained for the first time.

L132-144: Since the authors used commercial kits, it is not really necessary to explain the determination methods and therefore withdraw.

L146: How the authors fix the sample tissue at first? using formalin or buffered formalin and how?

L149: Considering that alcohols and paraffins are immiscible, an intermediate solvent must be used that can be mixed with both.

L157: Why was universal primer not used for 16S and the authors designed a pair primer?

L175: Why the authors did not provide growth performance and feed utilization? Any reason? The aquaculturists are enthusiastic to know the changes in weight gain and FCR values after 52 days feeding trial.

Why the results combined with discussion? It is optional or it is a journal’s rule? If it is optional, please separate them in the next round.

Please clarify all the abbreviations and treatments (PP, PP1, PP2, etc) in the captions and/or footnote of all tables and Figs.

Table 1: The proximate composition of some important ingredients (especially fishmeal, Yeast powder, and Soybean meal) should be provided in the footnote. Yeast powder for what reason was added?

Table 1: Since the authors used soybean oil (contain the most considerable amount of a natural source of lecithin), why they add lecithin at 1%?

Table 1: Please calculate and present gross energy for the diet.

Table 1: do you mean wheat flour by noticing only flour in the table?

Table 1: Mineral and vitamin premixes were calculated in 1 kg premix or 1 kg feed?

Critical comment: L200-204: Finally how or in which mechanism of action(s) PP can promote intestinal digestive enzymes activity to improve digestive functions ?

L270-271: How can PP do this?

L289: why they were removed?

Fig. 4b: why there are some unclassified bacteria? It means there were no recorded in the NCBI for them?

L341: the richness of intestinal is a good or bad sign?

L369: why? May be Proteobacteria can utilize more polysaccharides?

L371: in terms of Cyanobacteria, it is a good or bad sign?

L375: How? by which mechanism of action?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors accepted almost all of my suggestions. I have only some minor suggestions:

 line 50 - remove ", et al"

line 65: "marine" - no capital letter

line 182: replace "Duncun’s" by "Duncan's"

Tests for normal distribution is in supplementary material - OK, but at least indicate in the text which test was used

Figures and tables: authors decided to keep K, PP1, etc as the abbreviations for the treatments. OK, but then add in each figure and table caption the definition of each abbreviation, because as it is, the reader as to go back to methodology to find out which treatments are.

line 285: which PP levels were tested in grass carp?

line 289: I suggest "supplemental levels of PP did not improve further the growth of spotted sea bass compared to lower levels"

lines 292-293: add reference regarding this statement

line 300: remove the scientific name, it was placed previously in the text (line 285)

line 332: correct - "reached", "decreased"

No problems here

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The authors did their best to provide the revisions, however, there are some comments according to the response letter as follows:

-Again, the authors should provide the full terms of the abbriviated treatment names regarding the dose in the footnote or captions of tables and figs.

Based on the response provided by the authors, the purity of PP was only 50%, this is a question that what is other components in 50%? Since the purity of PP is low, can it interfer the results? What is your justification(s)?

Based on the growth performance results, what is the probable reason or reasons that the highest PP dose (PP5) could not improve the growth indices in your study? Any side effect for dietary PP in fish? Please supplement the discussion section in terms of low growth performance in PP5 diet, too.

Author Response

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Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Good luck

Author Response

Your valuable comments on the revision of this manuscript are greatly appreciated.