Assessment of Postbiotic, Mundticin-like Substance EM 41/3 Application in Broiler Rabbits

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article contains a considerable amount of data, but it is poorly structured and arranged. There is inconsistency throughout. Some studies have too small a sample size to draw conclusions (n=4). The methodological part is quite unclear, with some studies left undescribed. It is ambiguous whether the sample is representative of the group or the entire study. Similar to the results section, there is a lack of consistency. Table 1 displays reliability, but it is only marked in one place. Although it is stated that the productivity of rabbits was analyzed, this information is not presented in the results section. Overall, the quality of the article is too low.

Author Response

First of all, I would like to thank so much for time and effort to read and try to assess a manuscript and giving me  chance to improve it.

Rev.1 -in all colours I think the manuscript was improved and you can also find here some responses, notes and others directly in the text of mns.

The reviewer mention that for some analyses is small n used. Experimental work even on farm does not allowed to slaughter more animals (from our acceptation organ), so we used for that or those parameters n=4 from groups EG and CG to have orientation in parameter which also was used in other experiments with different substances and several repetitions as responsible and validated.

There was not mentioned that productivity was analyzed (in introduction), it was used only in introduction to explain why broiler rabbits are a good model animals that they have good productivity. So it was not meaning as a searched parameter nor result.

Some methods describing was improved

Reviewer 2 Report

Comments and Suggestions for Authors

Assessment of Postbiotic, Mundticin-like substance EM 41/3 2 application in broiler rabbits

General comments:

Research on the use of postbiotics in animal production and health is a current topic. The authors have put great effort into their research. However, important improvements must be made before the manuscript can be considered for publication.

First, it is imperative to clearly state the objective and add a hypothesis. This will enable the understanding of the materials and methods, and consequently facilitate the writing of the discussion and conclusions.

Although the manuscript is understandable, it is important to check the spelling and grammar, since it still contains several elements to improve.

In the Material and Methods section, it is necessary to clearly define the “Control” and the effects of treatments (postbiotics only or also the administration period of 21 or 42 days). Additionally, this must be included in the statistical analysis. Maybe the experiment is 2 x 2 factorial?

Once the issue of the treatments and statistical analysis were solved, the tables and results should be improved since the use of literals to identify the differences between means of treatment.

 

Specific comments:

Lines 62-66. It is necessary to state precisely what the objective and hypothesis of the study are.

Lines 114-118. Join the two sentences to obtain a single paragraph.

Line 121. Check and correct the term “Fo”

Line 121 to 133. Clarify how and with what frequency fecal sampling was performed for microbiota analysis.

Line 138 and 148. It is not clear whether 8 animals per treatment or 8 animals in total were blood sampled for both treatments. In any case, it should be clarified.

Line 151. Clarify how and with what frequency sampling for biochemistry was carried out.

162-165. In what substrate and at what time was the hydrolytic activity measured?

Line 168. Includes the mathematical calculation.

Lines 176-180. Separate the statistical analysis into a different section. Include in the analysis description which treatment effects were used. In its description, the authors mention a one-way ANOVA analysis, but in the results, the authors mention treatment effects (postbiotic), but also the sampling day. The authors should check this since the experimental design does not correspond to the statistical model and the results shown in the Tables. As presented in the results, there are not two treatments but there would be 5 treatments: 1) Control, 2) Day 21/EG, 3) Day 21/CG, 4) Day 42/EG, 5) Day 42/CG.

Line 185. Check and correct the term “significantly.”

Lines 183 - 297. Two problems. The authors did not declare a hypothesis, and the experimental design was not aligned with what is shown in the Tables. Then, the results are difficult to understand and follow. In addition, the wording of the results must be reviewed and corrected. I suggest you include only the statistically significant differences, eliminating any mention of mathematical differences.

Lines 359-362. Concerning the text: “Regarding the meat quality… are alltogether in association” the author must clarify how is this related to the postbiotic effect. Please discuss reports by other authors.

Line 362. Check and correct the term “alltogether”.

Lines 368-374. Concerning the text: “The indigenous intestinal microbiota… profile of bred animals” must be supported by previous studies.

Comments on the Quality of English Language

It is recommended to carry out a general review and correction of spelling and grammar.

Author Response

First of all, I would like to thank so much for time and effort to read and try to assess a manuscript and giving me  chance to improve it.

Rev. 2-in red or violet

Abstract was improved

Lines 62-63 (I dont know if it is in these lines now but main idea and objectives/aims are done there (in red/ in violet).

I think aim/objective was clearly involved now in introduction (it is in violet  and red because it is response also for rev. 3): and also main idea (it is not hypothesis but suppose)

R: Therefore, the aim of this study was to test effect/functionality of MLS EM 41/3 in broiler rabbits with focus on the following parameters: microbiota, PA as non-specific immunity parameter, glutathione-peroxidase activity (GPx) to assess oxidative stress, growth parameters, biochemistry in blood serum, jejunal morphology, organic acids in caecal chyme, fecal enzymatic activity, and meat quality. Our idea was to find if this new one enterocin can have similar effect in rabbits as previously detected and tested enterocins characterized in our laboratory. In addition, these aims have been conducted to secure rabbits health as food-derived animals and their meat as functional food. And the aim was also to test MLS EM 41/3 in broiler rabbit animal model before its use in horses.

Line 362 ........the term altogether was changed in... are associated paramaters

Lines 114-118 join in one paragraph. It was done:

 Musculus longissimus thoracis and lumborum (MLTL) was separated by removing skin, connective tissue, chilled and stored at 4 ˚C for 24 h until analysis. Rabbits were also regularly weighted. Rabbits blood (vena auricularis) was sampled into Eppendorf tubes (with and without heparin according to parameter analyzed) at day 0/1, 21 and 42.

Lines 121-133, frequency of sampling for microbiota analyses...it was involved in part of Experimental Design and Sampling but it can be involved again also in this part: To count microbiota in feces, sampling was provided at day 0/1 (before MLS EM 41/3 application), then at day 21 (after 3 weeks of MLS application) and finally at day 42 (3 weeks of MLS cessation). In case of caecum and appendix, sampling was performed at day 21 and 42.

Line 121...for microbiota enumeration.....

Lines 138 and 148: Sampling was performed at day 0/1 (n=8), at day 21 (n=8), and at day 42 (n=8) meaning mixture blood  from all animals from each one group as from EG so also from CG  to have mixture from all animals-  8 blood samples from each one group and each one sampling. It is explained in the text.

Line 151.... GPx (glutathione-peroxidase) activity (n=8, meaning at the start of experiment, at day 21 and at day 42, mixture blood samples of all animals from each group)

Lines 162-165 in original....  The degradation of the following substrates was measured: cellulose, starch, inulin, pectin, xylan).

Lines 176-180..statistical analysis....... It was not 5 treatment, there were CG and EG at 0/1, at 21 and at 42 and mixrure of samples, so I think this is Ok.

 2.6. Statistical analysis: Statistic analysis of treatment was analyzed using one-way analysis variance (ANOVA) with Tukey post hoc test. Data are expressed as means and standard deviation SD of the mean. Different superscript indicated significant difference p<0.05. Statistical analyses were performed by the using GraphPad Prism version 6.0 (San Diego, CA, USA).

Original line 168 (now changed), response: ADG can be calculated by taking the amount of weight an animal has gained since the last weight and dividing the weight by the number of days since that last weight. FCR= Feed given / Animal weight gain. In other words, the F.C.R. is the mathematical relationship between the input of the feed that has been fed and the weight gain of a population.

Line 185...significantly was revised

Lines 359-362, now 383..... As we don’t have exact explanation for influencing the meat quality using postbiotics, our hypothesis can be based only on idea of total postbiotic influence (MLS EM 41/3) on e.g. animal immunity.

Lines 368-374, now 394..... it was added-supported

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, MLS EM 41/3 was used as a postbiotic, and its effect on rabbit health status was studied with microbiota analyses, phagocytic activity analysis, glutathione-peroxidase evaluation, and biochemistry in blood serum, etc. This study is much meaningful for the cultivation of healthy food-derived animal with environmentally friendly method. Some questions in the manuscript need to be improved.

 

1. The abstract can not reflect what you have done in this study, and the significance of this study. Besides, there are some sentences that don’t made sense. The authors should reorganize it.
2. Routinely, its should be stated what will you study in this research and why you choose this topic. These information can be taken as the background of this study. The authors have made great effort, but they failed to give enough information on the research status of MLS EM 41/3. Moreover, they have include some information irrelevant with the topic. The authors should reorganize these information to make the introduction clear and concise.
3. As described in 2.1, inhibitory activity was tested with precipitate re-suspended in minimal volume of phosphate buffer. The concentration of the MLS EM 41/3 was not deleared. The inhibitory activity obtained here can not be used for further study,and can not be repeated by others. Thus, it cannot meet the requirement of academic papers.
4. The logical connectives is often misused,and it makes the results hard to read and understand.
5. L205 “They were not reduced.” What are they?
6. There are few references in recent years. It is suggested that the authors search for relevant research results in recent years and update them.

 

Comments on the Quality of English Language

Extensive editing of English language required. 

Author Response

First of all, I would like to thank so much for time and effort to read and try to assess a manuscript and giving me  chance to improve it.

Rev. 3-in violet

1. Abstract was improved
2. More information for MLS: Mundticin-like substance (MLS) EM 41/3 is thermo-stable substance of proteinaceous character. It is produced by horse-derived strain Enterococcus mundtii EM 41/3. This MLS producer strain was isolated from Slovak horse breed Norik from Muráň. It possess gene for Enterocin P and gene for Mundticin KS [6]. Under in vitro conditions MLS EM 41/3 has shown a broad antimicrobial (inhibitory) spectrum inhibiting also growth of listeriae and staphylococci with activity up to 800 AU/mL [6]. Based on origin of MLS EM 41/3 producer strain, MLS EM 41/3 has been aimed predominantly for use in horse breeding.

It is continually studied and trying to be completely purified.

I think also Introduction was more explained regarding the aim of this work.

 Therefore, the aim of this study was to test effect/functionality of MLS EM 41/3 in broiler rab rabbits with focus on the following parameters: microbiota, PA as non-specific immunity parameter, glutathione-peroxidase activity (GPx) to assess oxidative stress, growth parameters, biochemistry in blood serum, jejunal morphology, organic acids in caecal chyme, fecal enzymatic activity, and meat quality. These aims have been conducted to secure rabbits health as food-derived animals and their meat as functional food. And the aim was also to test MLS EM 41/3 in broiler rabbit animal model before its use in horses.

4. The logical connectives is often misused, and it makes the results hard to read and understand.

R: I again tried to improve achieved results. Mentioned logical connectives which were not understood by Rev. are not indicated here, so I am not sure if I know what she/he means but the others Rev. mentioned that it was understanable. But for each case, I changed and/or I think improved that results presentation.

3. I dont understand your statement. Inhibitory activity of substance has to be checked against principal indicator to know how active substance is. So it was introduced and indicated. So with this real activity MLS EM 41/3 was used in experiment and it is validated according to many others previous experiments based on basic in vitro results and experimented in animals. So, please maybe you work with bacteriocins application in animals but I cannot agree with this your argument. Concentration (inhibitory activity) can be used also by others when they will have our strain and protocol for MLS reaching and also indicator to test achieved activity.
4. They were not reduced. What are they?

R:  Coliform bacteria and amylolytic streptococci were not reduced in appendix.

In the text it is in red because I revised the text. So, I used red not violet here for Rev. 3.

6. I dont understand very well this note in point 6. We dont only upgraded our results. We tested in our team and in our lab characterized bacteriocins mostly enterocins repeatedly in different experiments using broiler rabbits which (those enterocins) are original (not commercial) and now new one enterocin -mundticin was tested, so it is not upgrade but new information and even as pre-experiment for use its possible use in other animal species. It is also possible to compare functionality of different enterocins/bacteriocins.

I am often in face of referees to use recent references, so it differs from referee to referee but to say that it is only upgrade is not true. It is original, new research again from our team.

English was checked but final check could be done again only after final acceptation of mns. Because it not has sense to do final language check if one more revision will be e.g. requested /expected before acceptation. Because language check is paid and to do it for several time it does not give me sense. So, if it will be necessary for final check language it will be definitely done.

Reviewer 4 Report

Comments and Suggestions for Authors

Lines 106-107 indicate what concentration of Mundticin-Like Substance (MLS) EM 41/3 was administered in drinking water

Justify the paragraphs of the lines 219-242 and 255-283

Author Response

First of all, I would like to thank so much for time and effort to read and try to assess a manuscript and giving me  chance to improve it.

In general, I think the manuscript was improved.

Lines 106-107 indicate what conc. of Mundticin-like substance MLS EM41/3 was administered in drinking water...it was add.

R: Animals were administered with Mundticin-like substance (MLS) EM 41/3 in drinking water (activity concentration 25 600 AU/mL...........

Justify paragraphs of the lines 2019-242 and 255-283:

R:It was checked, justified, improved and/or revised. You can check directly in the text (in bleu).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Corrections/adjustments in the methodological part and other sections are acceptable.

Author Response

Thanks for reviewing. Controlling notes, now I did not find any special comments from you in your review. So I cannot response pointly, only to say that manuscript was checked again and some parts were again revised and improved and/or changed involving final language check.

Reviewer 2 Report

Comments and Suggestions for Authors

Assessment of Postbiotic, Mundticin-like substance EM 41/3 2 application in broiler rabbits

General comments:

The authors have improved the manuscript significantly. However, two issues were not solved:

1) The hypothesis was not clearly stated yet. This is a scientific paper. Please include the hypothesis, not the main idea.

2) The authors mention that they have only two treatments: CG and EG. However, sampling was performed at day 0/1 (n=8), at day 21 (n=8), and at day 42 (n=8). So, the authors are using a two-way ANOVA (treatment and sampling days) with repeated measures (0, 21, and 42 days) on the “sampling days” factor. However, although the authors clearly state this in materials and methods, their statistical analysis and the way of presenting the results in the results tables are very different.

Comments on the Quality of English Language

None

Author Response

Thanks again that you assessed manuscript and here are explanations for two points: in red are changes

1. Regarding the hypothesis, I think that now it is better highlighted what was motive and hypothesis.

Resulting from our previous experiments using broiler rabbits and e.g. postbiotics characterized at our laboratory (Enterocin M, Durancin ED 26E/7) and/or their producer strains we have hypothesized that MLS EM 41/3 would have beneficial effect in animals without side effects [7-10].

Based on previous achievements with our enterocins and hypothesis formerly indicated, the study aim was to find if this new one enterocin can have similar effect in rabbits as previously detected and tested enterocins. In addition, these aims have been conducted to secure rabbits health as food-derived animals and their meat as functional food. And as formerly mentioned, the aim was also to test MLS EM 41/3 in broiler rabbit animal model before its use in horses.

2. Statistical
3. We dont have two treatments, we have one treatment -aim meaning to follow effect of MLS EM 41/3 after its 3 weeks application and then after 3 weeks cessation (and backround) and we have control group and experimental group which we compare. In some cases also other groups were compared, and maybe there are description of comparison.  
4. Thanks for your kindness