Effects of Dietary Supplementation of Humic Substances on Production Parameters, Immune Status and Gut Microbiota of Laying Hens
, Viera Karaffová 2,*, Boris Semjon 3, Pavel Naď 4, Jana Koščová 1, Martin Bartkovský 3, Andrej Makiš 3, Lukáš Bujňák 4, Jozef Nagy 3, Jana Mojžišová 5 and Slavomír Marcinčák 3,*Round 1
Reviewer 1 Report
Good work addressing comments
Reviewer 2 Report
none
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The work is prepared quite diligently, I have no major remarks. However, I have a few observations:
- To assess the laying performance more accurately, studies with a larger number of laying hens should be carried out. In this case there were only 30 hens in the group.
- Line 343: Incorrect indication of the HS insertion rate used in the investigations.
- The reasons for the increase in B lymphocytes and decreased the proportion of T lymphocytes should be more widely commented.
Reviewer 2 Report
this MS presents a trail of lying hens with oral supplement of humic substance, and results showed humic exert benificials in lying rate, egg quality, immune and intestine microbiota. It is at least meaningful to use humic as functional additive. thus, I suggest minor revision.
comments:
abstract:
- line 20: in this study was …., it is wrong grammar
- lin2 27: there was observed, wrong grammar
introduction:
first paragraph:
it is no needed to describe the characteristics other than in relation to animal nutrition.
Methods:
Why only one dosage of supplement was used?
Why not to detect IgA content in intestine by ELISA？
Reviewer 3 Report
Overall the manuscript needs an extensive revision on materials and methods and diet composition details. There are 2 main concerns.
- Replicates per treatment are low (n=3) how authors ensured there was enough statistical power?
- The authors described taking several measurements daily for 29 weeks, however, only final week 29 data is presented. I encourage authors to add figures with week performance progress and arrange data to know what happened during the previous 29 weeks. Should a repeated measures analysis is more appropriate for this type of data over time??
Please find attached further comments in the PDF file.
Comments for author File: 
Comments.pdf
Reviewer 4 Report
General comments
Throughout the paper, it is not possible to speak of immunomodulation of the cellular or humoral response, but only of an increase in the percentage of immunocompetent cells with expression of a specific CD molecule or an increase in the expression of a given gene. The authors have shown too few connections between these parameters. The word ‘immunomodulation’ should be changed throughout the manuscript.
The Discussion proposed by the authors is laconic and does not provide an interpretation of the results. Unfortunately, while reading the article submitted for review I have the impression that it is very similar to the article “The effect of humic substances on gut microbiota and immune response of broilers”, published in Food and Agricultural Immunology. For this reason the authors absolutely must revise the entire Discussion.
Introduction
39 – What microelements?
40-41 – In what forms and in what concentrations was it used in poultry? Provide details.
74 – What subpopulations of lymphocytes were tested?
Materials and methods
93 – Explain why the amount of 0.5% HS was chosen.
104 – How were health parameters monitored?
105 – Only 10 birds were slaughtered from two groups (i.e. 5 individuals from each group)? If so, this is in no way a statistically significant group for evaluation of the parameters.
158-162 – Delete. The principle of the test is well-known and described in the package insert.
165 – To assess humoral activity following the use of HS, the levels of lymphocytes with expression of, for example, Bu-1-a and CD79a should be determined, which was not done in this study. The CD8 alpha receptor co-occurs on lymphocytes with CD4 expression, whereas CD8 alpha/beta expression is found on cytotoxic Tc lymphocytes. Therefore is it not worth considering the use of a different antibody?
I recommend reading the paper “Chicken CD4, CD8ab, and CD8aaT Cell Co-Receptor Molecules” by M. Luhtala and including some of the information in your Discussion.
172- How did you test the concentrations of mononuclear cells?
177-179 – Delete
197 – What species of Gram-negative bacteria of the family Enterobacteriaceae and what species of LAB were tested? How were they classified and identified?
Discussion
326-328 – Delete this sentence.
341-356 – There is no actual discussion of the effect of HS on the immune response. The discussion lacks clarity.
Reviewer 5 Report
Page 2, line 89: to be replaced by "birds", "layers" or "poultry"
page 2, line 94: I do not recognize this product! maybe you meant "sunflower meal"!
page 2 line 94: I think it is soybean meal!
page 2, line 96: replace with oil
page 2, line 101: replace the word because it repeats itself!
page 3, line 103: change to "hen" or "layer"
page 3, line 105: 5 birds / group is not a large enough number for a statistical analysis! Moreover, what would be the criterion for random selection of the 5 chickens / lot from the 3 cages /group?
page 3, line 105: replace with "birds"
page 3, line 109, table 1: It is necessary to present the structure of the diet and the premix depending on the growing phase of the birds. to be completed from the guide!!!!!!!
page 3, line 114: replace with group!!!!
page 3, line 115: to be replaced!
page 3, lines 119-121: Which would be the explanation why eggs collected at the end of the experiment were not analyzed immediately after collecting, referring, of course, to egg shell, the analysis of minerals being done after 14 days of storage in the refrigerator? What would be the reason why the eggshells were, after 14 days, frozen at -20 °C before the actual analysis?
page 5, line 211: I suggest you to complete table 4 or to present in a graph the evolution of the production parameters from week 17 to week. 29!
page 5, line 215: as in table 5
page 6, table 4: "H" group or "HS" group
page 6, line 216: It is not very clear, the experimental group is called "H" or "HS" group. Moreover, from the data presented in the material and the method it is understood that the experiment was carried out on laying hens Lohman Brown 17-29 weeks. In the footer of the table, the experimental group "H" is assigned "broilers" and not "layers". Please review the footnotes for all the tables in the manuscript!
page 6, table 5: Please mark the differences between the groups with superscripts!
page 6, lines 232-233: the legend is positioned below the figure! Check all the figures in the paper!
