Immune Response and Fatty Acid Profile of Eggs from Laying Hens Fed Fermented Feed Rich in Polyunsaturated Fatty Acids

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript, # Fermentation -1591686, “Immune response and fatty acid profile of eggs from laying  hens fed fermented feed rich in polyunsaturated fatty acids” by Viera Karaffová  et al. investigated the effect of fermented feed on the selected immune relevant parameters in laying hens and fatty acid profile of produced eggs.

 General comments:

First:

I think that the article does not fit in with the aims and scope of the website.

https://0-www-mdpi-com.brum.beds.ac.uk/journal/fermentation/about

for example:

Fermentation process and product development

Strain improvement

Bioprocess and metabolic engineering

Fermentation food and beverages

Scale up of fermentation processes

Downstream processing of fermentation products ….

The authors do not describe the fermentation process because it has been previously published, but  investigated the effect of fermented feed on the selected immune relevant parameters in laying hens and fatty acid profile of produced eggs. I think this article should send to another journals not that this.

Second:

What was fermented - all control mixtures or just wheat bran? If wheat bran- that I don't understand this project and I my opinion this experiment had the wrong assumptions. Why authors replaced control mixtures on 10 or 15% level fermented feeds?  I think it was fermented wheat bran, but 10 or 15% level this feeds has another’s nutritional value than all mixtures.

 I think, this research not conducted correctly.

Major comments:

Another point deals with the inconsistency between title and abstract. You speak of immune response and fatty acid profile of eggs and then start the abstract with type of diet. I would identify the hypothesis - I didn't see in all text.

Material and methods: The authors should provide the composition  and nutritional value of the mixtures in the table, not only fatty acid profile in laying hens of fermented feed and mixtures.

I don't feel qualified to judge about the English language and style, but I seen many place where are extensive  changes required.

Thank you.

Author Response

Answers to reviewer:

In the first place we would like to express our thanks for stimulating suggestions. We tried to correct the manuscript based on reviewer´s demands and/or explain some points. All corrections in the manuscript are marked with yellow background.

General comments:

First:

I think that the article does not fit in with the aims and scope of the website.

The authors do not describe the fermentation process because it has been previously published, but  investigated the effect of fermented feed on the selected immune relevant parameters in laying hens and fatty acid profile of produced eggs. I think this article should send to another journals not that this.

Before sending the article to the Fermentation, we contacted the editors and consulted with them on the suitability of our manuscript for their journal. Based on their recommendation, we sent a manuscript stating that it focuses on the direct impact of the fermented product on animal health / production.

We also described the fermentation process in more detail.

Second:

What was fermented - all control mixtures or just wheat bran? If wheat bran- that I don't understand this project and I my opinion this experiment had the wrong assumptions. Why authors replaced control mixtures on 10 or 15% level fermented feeds?  I think it was fermented wheat bran, but 10 or 15% level this feeds has another’s nutritional value than all mixtures. I think, this research not conducted correctly.

Fermentation was only presented on the wheat bran. The main idea of the research is the use of agricultural waste for feed purposes, which is particularly advantageous from the ecological and economic point of view. In addition, filamentous fungi can produce various biogenic substances (such as PUFA or carotene) on plant-based wastes, which will increase the quality of the carcass / animal product, and can even be considered a functional food for humans. Fermentation of all mixtures for large poultry farming is economically unprofitable.

We combined 10 and 15% of fermented product with commercial feed mixtures according to our previous research (Kovalík et al. 2018; Bartkovský et al. 2020). We decided to use 10  and 15 % of FP addition according to previous results and fungal strains potential.

Kovalík, P., Mačanga, J., Klempová, T., Popelka, P., Marcinčáková, D., Mellen, M., Bartkovský, M., Maskaľová, I., Čertík, M., Marcinčák, S. Effect of feeding of 5% prefermented cereal-based bioproduct enriched with γ-linolenic acid on production indicators, chemical composition, fatty acid profile and lipid oxidation of broiler meat. In Italian Journal of Animal Science. ISSN 1594-4077, 2018, vol. 17, no. 2, p. 208-417.

Bartkovský, M., Mudroňová, D., Marcinčáková, D., Klempová, T., Sesztáková, E., Maskaľová, I.,  Karaffová, V., Jaďuttová, I., Čertík, M., Hudák, M., Marcinčák, S. Effect of fungal solid-state fermented product enriched with gamma-linolenic acid and beta-carotene on blood biochemistry and immunology of broiler chickens. In Polish Journal of Veterinary Sciences. ISSN 1505-1773. vol. 23, no. 2 (2020), p. 247-254.

Major comments:

Another point deals with the inconsistency between title and abstract. You speak of immune response and fatty acid profile of eggs and then start the abstract with type of diet. I would identify the hypothesis - I didn't see in all text.

The main goal of our research is to change the PUFAs profile of animal products by feeding cheap biotechnologically prepared feed with the required PUFAs composition (e.g. n-3 PUFA or GLA fortified foods). As confirmed by research, PUFAs fed to animals are converted into animal products and it is thus possible to enrich food for humans, but at the same time a change in the composition of PUFAs in animal food affects their health, especially the immune response. Therefore we started Abstract and also Introduction with importance of PUFAs composition in diets.

The hypothesis is given at the end of Introductrion:

“A potential improvement can be brought about by changing the fatty acid profile of animal products of slaughter animals by adjusting their diet. For this, it is possible to use feed biotechnologically enriched with specific fatty acids, the consumption of which can transfer these fatty acids to slaughter products - meat and eggs in the case of poultry. In this way, it is possible to improve the ratio of n-3 and n-6 PUFAs consumed and, in addition, to positively affect animal health, in particular their immune response.”

Material and methods: The authors should provide the composition  and nutritional value of the mixtures in the table, not only fatty acid profile in laying hens of fermented feed and mixtures.

Composition of feed mixtures (Table 1) has been added to the manuscript.

Reviewer 2 Report

An interesting work where the effect of supplementing a fermented feed rich in polyunsaturated fatty acids on immune response and egg quality of hens is presented.

The work has potential interest and application for producers and consumers .

However, there are issues which need to be corrected/clarified to improve the manuscript. Main comment below

.- authors need to clarify sample size, experimental unit (true replicates).

.- for many variables it is not clear how samples were taken, when it was take, etc.

.- details of diet composition and intake by hens are needed. this will allow  author, reader, producers to estimate the intake of polyunsaturared fatty acids needed to achieve such results. Also, will clarify the contribution (%) of the fermented food in the overall supply of u-FA

.- the discussion must include a section related with intake

Further detailed comments are in the attached file -

Comments for author File: Comments.pdf

Author Response

 Answers to reviewer:

In the first place we would like to express our thanks for stimulating suggestions. We tried to correct the manuscript based on reviewer´s demands and/or explain some points. All corrections in the manuscript are marked with yellow background.

Reviewer:

However, there are issues which need to be corrected/clarified to improve the manuscript. Main comment below

.- authors need to clarify sample size, experimental unit (true replicates).

.- for many variables it is not clear how samples were taken, when it was take, etc.

.- details of diet composition and intake by hens are needed. this will allow  author, reader, producers to estimate the intake of polyunsaturared fatty acids needed to achieve such results. Also, will clarify the contribution (%) of the fermented food in the overall supply of u-FA

.- the discussion must include a section related with intake

Comments

A table with detailed nutrient (chemical) composition must be included.

Composition of feed mixtures (Table 1) has been added to the manuscript.

What nutrient standard was employed as reference?

As a nutrient standard a commercial feed for laying hens was used for the experiment. Standard values for feed were in accordance with Act no. 271/2005 Coll. (Law on the production, placing on the market and use of feed).

How many samples for FA profiles of egg yolk and feed mixtures were analysed? How samples were collected and preserved?

The information have been added to the manuscript (section 2.3).

Describe the sample, sample preparation and number of samples for relative gene expression analysis.

The information have been added to the manuscript (section 2.4).

How samples for phagocytic activity were collected and preserved? How many samples?

The information have been added to the manuscript (section 2.5).

Microbiological screening – how many samples?

The information has been added to the manuscript (section 2.7).

It is important to present and discuss the intake levels obtained /needed to achieve the positive results presented.

In the experiment, we used the feed ration recommended by the Lohman brown classic management guide, which even with the addition of 10% fermented product proved to be sufficient to significantly increase the proportion of n-3 PUFAs. This knowledge was added to the Discussion. As the experiment was focused on egg quality and hen health, we did not monitor the exact feed intake.

Reviewer 3 Report

The aim of the research was to evaluation immune response and fatty acid profile of eggs from laying hens fed fermented feed rich in polyunsaturated fatty acids. The Introduction chapter provides an overview of the current knowledge on the topic of the article. The number of hens is small but sufficient. The article lacks information on the number of immune response tags and the number of eggs assessed. The research methods used are correct. In the Material and methods chapter, there is no information on the hens' housing conditions and the content of CP and MJ ME, the form of the feed mixture. The Results chapter requires the some places revision. The discussion was conducted well and comprehensively. The Conclusions do not describe the influence of the fermented feed on immune response. The article needs to be supplemented and corrected before being printed in the Fermentation journal. The list of recommended changes is provided below:

General comments

Please prepare the article in accordance with the instructions for authors

For significance please use „p” lowercase in italic

The titles and legends for figures should be below the figures and not above the figures

For a range of pages in References use the long character "" with insert symbol (tab symbol)

Detailed comments

L69 use the phrase "The purpose of the research was ..." when formulating the purpose of the research

L81 + please add information about the type of building (closed building, without windows?), environmental parameters (temperature, humidity. length, intensity, color of light), bird density, content of CP, MJ ME, calcium, phosphorus, Liz, Met in the feed mixture (maybe from the label), form (fine?) of feed mixture

L110 check correctness of KH2PO4 writing

L124 FP in full

The headings of the figures 6 below the figures, not above the figures

Markings of significance are not very clear, there are no comparisons between the F10 and F15 groups

I propose two levels a,b for p  0.05 and A,B for p  0.01

No significance markings for Figure 4a, group F10

No significance determinations were made for Figures 5b and 5c

No significance markings for Figure 6b, small intestine

L277 please provide the formula for gondoic acid

L290 use „p  0.05” instead of p  0.05, wrong description

L281 GLA also?

L400 FP in full

L404 add a description of the fermented feed impact on immune response

L473 dot after "Rep."

Comments for author File: Comments.doc

Author Response

Answers to reviewer:

In the first place we would like to express our thanks for stimulating suggestions. We tried to correct the manuscript based on reviewer´s demands and/or explain some points. All corrections in the manuscript are marked with yellow background.

Reviewer:

The aim of the research was to evaluation immune response and fatty acid profile of eggs from laying hens fed fermented feed rich in polyunsaturated fatty acids. The Introduction chapter provides an overview of the current knowledge on the topic of the article. The number of hens is small but sufficient. The article lacks information on the number of immune response tags and the number of eggs assessed. The research methods used are correct. In the Material and methods chapter, there is no information on the hens' housing conditions and the content of CP and MJ ME, the form of the feed mixture. The Results chapter requires the some places revision. The discussion was conducted well and comprehensively. The Conclusions do not describe the influence of the fermented feed on immune response. The article needs to be supplemented and corrected before being printed in the Fermentation journal. The list of recommended changes is provided below:

 All immunological and microbiological analyses were performed in all hens included in the experiment, it means in each group n = 10. These information have been added to the manuscript.

General comments

Please prepare the article in accordance with the instructions for authors.  

A journal template was used to prepare the manuscript and was edited according to the instructions on the journal website.

For significance please use „p” lowercase in italic

All "P" labels have been changed to lowercase.

The titles and legends for figures should be below the figures and not above the figures

The titles and figues were corrected.

For a range of pages in References use the long character "-" with insert symbol (tab symbol).

Long character "-" was used in all references.

Detailed comments

L69 use the phrase "The purpose of the research was ..." when formulating the purpose of the research

The sentence has been corrected.

L81 + please add information about the type of building (closed building, without windows?), environmental parameters (temperature, humidity. length, intensity, color of light), bird density, content of CP, MJ ME, calcium, phosphorus, Liz, Met in the feed mixture (maybe from the label), form (fine?) of feed mixture.

Information on housing, lighting as well as feed composition (Table 1) was added to section 2.1 of Materials and methods.

L110 check correctness of KH2PO4 writing.

„KH2PO4“ was changed to „KH₂PO₄“.

L124 FP in full.

FP was re-write to fermented product.

The headings of the figures 6 below the figures, not above the figures.

All headings of the figures were corrected.

Markings of significance are not very clear, there are no comparisons between the F10 and F15 groups. I propose two levels a,b for p < 0.05 and A,B for p < 0.01.

For the statistical analysis of differences between individual groups was used  Tukey's post-hoc test which compares all pairs of groups. It means, the F10 and F15 groups were  compared, because we also wanted to see differences in the administration of different concentrations.

We commonly use 3 levels of probability: 95%, 99% and 99.9%, which are marked with different superscripts, i.e.:

The superscripts are clearly marked, as:

if p<0.05^ab - it does mean that “a” is higher value than “b” value at the level of p < 0.05

if p<0.01^ac - it does mean that “a” is higher value than “c” value at the level of p < 0.01

if p<0.001^ad it does mean that “a” is higher value than “d” value at the level of p < 0.001

However, it may happen that some value is higher (e.g. “a”) than another at a certain level (e.g. “b”) and at the same time is lower (e.g. “c”) than other value. In this case, superscript in the graph for this group would be marked as “ac”.

This type of statistical labeling is commonly used in scientific publications if the number of monitored groups is not high (in our case only 3). But yes, indeed, in some cases it can be more difficult readable (e.g. Fig 2, where groups F10 and F15 differ at a significance level of p < 0.01, F10 and control at a significance level of p < 0.05, and F15 and control at a significance level of p < 0.001).

No significance markings for Figure 4a, group F10.

Yes, it is correct, it is not marked, because there was no significant difference between F10 and F15 or F10 and control

No significance determinations were made for Figures 5b and 5c.

Yes, it is correct, because there were no statistical differences (in both cases ANOVA: p > 0.05) - mainly due to higher standard deviations.

No significance markings for Figure 6b, small intestine.

Yes, it is correct, because there were no significant differences.

L277 please provide the formula for gondoic acid.

Formula for gondoic acid was added.

L290 use „p > 0.05” instead of p < 0.05, wrong description.

It was corrected.

L281 GLA also?

Yes, also for GLA, please see details in the eighth line of Table 3.

L400 FP in full.

„FP“ was changed to „fermented product“.

L404 add a description of the fermented feed impact on immune response.

The description has been added to the conclusions.

L473 dot after "Rep." – dot was added after „Rep“

Round 2

Reviewer 2 Report

The authors have answered all queries from the previous round

no further comments