Growth Performance and Ruminal Fermentation in Lambs with Endoparasites and In Vitro Effect of Medicinal Plants

Round 1

Reviewer 1 Report (New Reviewer)

Agriculture 2600969 peer review

Growth performance and ruminal fermentation of lamb infected with gastrointestinal nematode (GIN) was investigated, and compared with the control. While, in vitro fermentation of rumen microbe from lamb infected and un-infected with GIN was studied by treated with plant bioactive compound sources.

The present manuscript composed from 2 different experiments which not total related. My first opinion is that the author could clearly separate part of Exp. I and Exp. II, both M&M, Result, and Discussion. Just final discussion that can be use result from Exp. I for Exp. II. If use wording in vivo and in vitro trial, the reader may misunderstand that it has similar treatment, but not.

The title has no word that saying about plant bioactive compound which is treatment in Exp. II.

There are some interaction effect between Substrate and Inoculum, these should be more statistical analysis to know how Substrate act in Con vs Infected

Lack of treatment comparison for Substrate effect, how to interpret the result when it significant. For example, NH3-N was significant affected by Substrate but don’t know significant between MH, Herb and chicory.

Author Response

Dear Rewiever 1,

              attached you can find revised manuscript D: agriculture-2600969, Growth performance and ruminal fermentation in lambs with endoparasites and in vitro effect of medicinal plants, by Klára Mikulová et al., for your consideration for publication in Agriculture.

Detailed responses to each point were clearly highlighted by red colour in revised manuscript and identified in the response letter as Revision note.

We are grateful for comments and hope our corrections and additions have improved the manuscript sufficiently for publication.

Yours sincerely,

Zora Váradyová, behalf all of the co-authors

 

Revision Note:

REVIEWER 1 (R1)

Comments and Suggestions for Authors

 

R1: My first opinion is that the author could clearly separate part of Exp. I and Exp. II, both M&M, Result, and Discussion. Just final discussion that can be use result from Exp. I for Exp. II.

AUTHORS: We separated part of Exp. I and Exp. II, both M&M, Result, and Discussion according to comments of R1.

R1: The title has no word that saying about plant bioactive compound which is treatment in Exp. II.

AUTHORS: The title was changed according to comments of R1: “Growth performance and ruminal fermentation in lambs with endoparasites and in vitro effect of medicinal plants”

R1: There are some interaction effect between Substrate and Inoculum, these should be more statistical analysis to know how Substrate act in Con vs Infected. Lack of treatment comparison for Substrate effect, how to interpret the result when it significant. For example, NH3-N was significant affected by Substrate but don’t know significant between MH, Herb and chicory.

AUTHORS: Individual differences (Table 5) were determined using Tukey’s multiple-comparison post-test and were considered to be significant at p < 0.05. Significance effects were added in to the Table 5, according to comments of R1.

 

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

The subject is interesting; however, some cases should be corrected/clarified. A moderate revision is recommended.

L82: Individually housed or in groups?

L195: What about the effect of "three times within three consecutive days" in the statistical model?

Table 1: Delete data on nitrogen. Provide EE concentration.

Table 2: The daily feed intake of two treatments should be reported.

Table 5: Different means should be specified using superscripts.

L237: Why were the results repeated in the discussion section? Avoid such an approach. Moreover, the discussion should be structured using suitable subheadings (In vivo and in vitro results should be discussed under different subheadings). In addition, it is better to discuss the effect of the animals (control vs. infected) and the effect of the plants separately. Finally, the description of the results should be improved.

L241: The DWG P value was 0.051. That is, there was a decreasing trend. Therefore, it is better to present the reasons.

L247-249: I do not agree with these explanations, because the BW and DWG differences between the two groups are large and close to significance. The decreasing trend can be clearly seen in the table.

In addition, the daily feed intake of animals must be reported. Sure.

 

The English language of the manuscript is understandable and appropriate. However, a few cases need to be controlled and corrected by the authors.

Author Response

Dear Rewiever 2,

              attached you can find revised manuscript D: agriculture-2600969, Growth performance and ruminal fermentation in lambs with endoparasites and in vitro effect of medicinal plants, by Klára Mikulová et al., for your consideration for publication in Agriculture.

Detailed responses to each point were clearly highlighted by red colour in revised manuscript and identified in the response letter as Revision note.

We are grateful for comments and hope our corrections and additions have improved the manuscript sufficiently for publication.

Yours sincerely,

Zora Váradyová, behalf all of the co-authors

Revision Note:

REVIEWER 2 (R2)

R2: L82: Individually housed or in groups?

Authors: L82: We added: “…were housed together in common stalls on…”

R2: L195: What about the effect of "three times within three consecutive days" in the statistical model?

Authors: L195: The fresh ruminal contents for in vitro experiment were collected from two lambs per group that were killed on days 48, 49, and 50 after infection (i.e., three times within three consecutive days)

R2: Table 1: Delete data on nitrogen. Provide EE concentration.

Authors: We removed the data of nitrogen from Table 1 according to R2 comments. I don't know what EE concentration means.

R2: Table 2: The daily feed intake of two treatments should be reported.

Authors: The daily feed intake of animals was added in Table 2.

R2: Table 5: Different means should be specified using superscripts.

Authors: Individual differences (Table 5) were determined using Tukey’s multiple-comparison post-test and were considered to be significant at p < 0.05. Significance effects were added (using superscript) in to the Table 5, according to comments.

R2: L237: Why were the results repeated in the discussion section? Avoid such an approach. Moreover, the discussion should be structured using suitable subheadings (In vivo and in vitro results should be discussed under different subheadings). In addition, it is better to discuss the effect of the animals (control vs. infected) and the effect of the plants separately. Finally, the description of the results should be improved.

Authors: The repeated results were removed from Discussion. We separated part of Experiment 1 and Experiment 2, both M&M, Result, and Discussion according to comments

R2: L241: The DWG P value was 0.051. That is, there was a decreasing trend. Therefore, it is better to present the reasons.

Authors: In the first paragraph in the Discussion section, we stated: "In the Experiment 1, growth performance expressed as DWG was relatively lower in GIN-infected lambs compared to control, but not significantly (72.6 vs. 130.1 g/day, Table 2)". We also present possible reasons for this trend and that is a parasitic infection, which is referred to the citation in the text.   

R2: L247-249: I do not agree with these explanations, because the BW and DWG differences between the two groups are large and close to significance. The decreasing trend can be clearly seen in the table. In addition, the daily feed intake of animals must be reported. Sure.

Authors: As stated in Materials and methods (section 2.2.1.) "Each animal was fed daily meadow hay (MH) ad libitum and 300 g dry matter (DM) of Mikrop ČOJ, a commercial concentrate." Large individual differences between animals were also noted when weighing animals. However, the daily feed intake of animals was not evaluated. It has been documented in the past that parasitic infection with H. contortus can often result in a period of reduced appetite, nausea, gastroenteritis, anaemia and poor nutritional absorption, which can lead to weight loss. We added in the Discussion: “In present experiment large individual differences between animals were also noted when we weighing animals. It has been documented in the past that parasitic infection with H. contortus can often result in a period of reduced appetite, nausea, gastroenteritis, anaemia and poor nutritional absorption, which can lead to weight loss [40].“

New reference was added:

[40] Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., Aguilar-Caballero, A.J., Cámara-Sarmiento, R., Alonso-Díaz, M.A., 2012. Nutritional manipulation of sheep and goats for the control of gastrointestinal nematodes under hot humid and subtropical conditions. Small Rumin. Res. 103, 28–40.

R2: Comments on the Quality of English Language

The English language of the manuscript is understandable and appropriate. However, a few cases need to be controlled and corrected by the authors.

Authors: The English have been revised throughout the whole manuscript by a native English language Editor. (Dr. William Blackhall, Global Biological Editing. www.globalbiologicalediting.com

 

Author Response File: Author Response.pdf

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

1- The experiment in vivo and in vitro are not clear. 

2. The presentation of results needs to be improve

3. The discussion is no clear. There was not citation in many points

4. The author said that colected abomasun contents but not showed the results

On the discussion section the english needs to be more clear

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

Thank you for sending the manuscript to me. This manuscript has two objectives, 1) to evaluate the effect of parasites infection on the performance and rumen microbiota of lambs, 2) to evaluate the effect of herbal plants on the rumen fermentation parameters and protozoan counts, in vitro. I do suggest keeping the result of In vivo experiment and removing the results of In vitro experiment as this experiment does not serve the In vivo experiment. The main purpose of the herbal plants is to disinfect the gut parasites and affect the rumen fermentation to decline the methanogensis; therefore, the authors had to try the herbal plants In vivo not In vitro. For example, the authors could have added two additional experimental groups to study the effect of herbal plants on the infected groups.

Title

The title of the manuscript does not cover the objectives of the manuscript, please change it.

Abstract

The abstracts need rewriting completely, to show the objectives and organize and clarify the objectives, experiments, results, and conclusion.

Line 27: what are the values of the fermentations? Do you mean the fermentation parameters?

Introduction

The hypotheses and objectives of the study are not clear in the introduction section.

Material and methods

Line 109-110: these lines refers that you used rumen inocula from control, infected, and herbal-treated animals, please clarify that.

Why did you add the worms to serum bottles, did you expect they will affect the fermentation, In vitro?

Also, do you expect that the worms survive in the fermentation medium?

Please clarify the fluorescence in situ hybridization technique that you used, and why didn’t you use the qPCR or NGS techniques instead of the hybridization?

 Results and discussion

Animal performance table: the authors should include initial body Wight, final body weight, and Average daily gain to clarify the effect of the infection.

 Could you explain the increase in CH4, the decline in propionic, and the increase in acetic in infected animals and please link that to the changes in animals’ body weight?

 

Please explain the increment in the xylanase in infected animals

the language of the manuscript needs extensive editing

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

This MS topic is interesting and suitable for consideration in agriculture. However, there are multiple concerns with the introduction, methodology, and results sections that require major revision. I have specific comments and suggestions for the MS improvement  as follows;

L2, L19 should be italic "in vivo" "in vitro''

l21 spell out the abbreviation "ISE" "MHCol"

L21 change "Two" to "Three" ?

L67-73 reword to be simplified & clearly objective

L62 spells out ISE, MHCol

L95 change "Two" to "Three" ?

L95 specifies the killing time of each day because microbes population and fermentation end product status changes during the before-after feeding 

L109 reword to clearly define a specific experimental design and treatment combination eg. T1= ?,.. T4=?

L117 a 3x2x3 ... not clear, a 2x3 factorial arrangement in CRD? if so, define what factor of the study. Replication?

L124 L124-128 move all methods and analysis related to in vitro section 2.3

L201 specify factor eg. 3(???) 2(??) 3(???)

L202 does not clearly define

L210 n=12?

L217 Table 3 1) Spell out  & specify the standard unit of all variables CH4, H2, SCFA 2) check 0.40 vs 0.49; is it significant at 0.001? 3)footnote wRC?

L225 Table 4 check all variables units in 10^5 cell/g wRC? Check all variable 

L236  1) check 3substratex2inoculumx3replicate=18 experimental unit(serum bottom) ? 2)provide statistical analysis, standard error of the mean (SEM) 3)CH4 unit? mM/gDM? or mM/L of TGP?

 

 

 

 

 

 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Thanks for responding the comments 

Please check the manuscript for language errors 

Author Response

Dear Reviewer 2,

in the attachment you can find the revised manuscript (Manuscript ID: agriculture-2538324-R2): Growth performance, nutrient composition and ruminal microbial fermentation associated with endoparasites of lambs, by Klára Mikulová, Daniel Petrič, Michaela Komáromyová, Dominika Batťányi, Martyna Kozłowska, Adam Cieslak, Sylwester Ślusarczyk, Marián Várady and Zora Váradyová.

Thank you very much for your constructive comments.

Yours sincerely,

Zora Váradyová, on behalf of all co-authors

Reviewer 3 Report

The article has serious flaws that led me to suggest rejection. Multiple concerns with the introduction, methodology, and results sections lead to the poor and unorganized discussion section. My specific comments are as follows; 

L66-75 does not clear objective and method design to solve the problems

L128-133, L212 The experimental design and statistical analysis was not agreed with either CRD or RCBD. The factor treatment and 3x2x3 arrangement are not clearly designed. 

L121 Methane was not analyzed using a Perkinelmer?

L226 Table 3 suggests to delate CH4, H2 data because it does not direct analyze and not agree with CH4 concentration.

L339 not cleared related to Table3.

 

 

 

 

Author Response

Dear Reviewer 3,

              in the attachment you can find the revised manuscript (Manuscript ID: agriculture-2538324-R2): Growth performance, nutrient composition and ruminal microbial fermentation associated with endoparasites of lambs, by Klára Mikulová, Daniel Petrič, Michaela Komáromyová, Dominika Batťányi, Martyna Kozłowska, Adam Cieslak, Sylwester Ślusarczyk, Marián Várady and Zora Váradyová.

Thank you very much for your constructive comments. We have carefully reorganized the manuscript according to the suggestions. Detailed responses to each point were clearly highlighted by red colour in revised manuscript and identified in the Revision note.

Yours sincerely,

Zora Váradyová, on behalf of all co-authors

Revision Note:

REVIEWER 3

Comments and Suggestions for Authors

The article has serious flaws that led me to suggest rejection. Multiple concerns with the introduction, methodology, and results sections lead to the poor and unorganized discussion section.

AUTHORS: We tried to eliminate all the shortcomings as best as possible.

L257-283: The Discussion has been properly reorganized under Results and Tables.

 

REVIEWER 3: L66-75 does not clear objective and method design to solve the problems

AUTHORS: L61-69: We changed the text as follows: “Little information is available on the effects of GI parasitism on ruminal fermentation we hypothesised that plants with antiparasitic properties would also influence the ruminal microbial communities of GIN-infected lambs in vitro. In addition, the use of mallow, chamomile, Jacob's ladder, wormwood, and chicory in the feed of ruminants provides high-quality roughage with a high content of crude proteins (from 140 to 160 g/kg dry matter) [16–18]. Therefore, we used the traditional medicinal plants mallow, chamomile, fumitory, wormwood, and chicory as substrate in fermentation in vitro based on their antiparasitic properties [13,19]. We simultaneously investigated lamb growth, nutrient composition and ruminal microbial fermentation associated with H. contortus.”

 

REVIEWER 3: L128-133, L212 The experimental design and statistical analysis was not agreed with either CRD or RCBD. The factor treatment and 3x2x3 arrangement are not clearly designed.

AUTHORS: L119-124: We removed the confusing sentence and tried to simplify the text of in vitro experimental design: “2.3.1. Experimental design Three replicates (three incubation serum bottles) were prepared for MH, Herbmix or chicory and each inoculum (CON and INF). The experiment consisted of fermentations of the three substrates in fermentations with two inocula (CON and INF) and repeated three times within three consecutive days (n = 3 × 3). At the same time, three replicate bottles were also used for the blank (inoculum, no substrate).”

L190: 2.6. Statistical Analysis: The incorrect interpretation has been removed from the text. We changed the text as follows: “Data on fermentation parameters and protozoan populations in vitro were analysed by two-way ANOVA (GraphPad Prism 9.2.0 (332) 2021; GraphPad Software, Inc., San Diego, USA). The model included effects for three substrates (MH, Herbmix and chicory), two inocula (CON and INF), and interactions between substrates and inocula.”

REVIEWER 3: L121 Methane was not analyzed using a Perkinelmer?

AUTHORS: L114-116: SCFA and methane in vitro were determined on Perkin Elmer Clarus 500 gas chromatograph.

REVIEWER 3: L226 Table 3 suggests to delate CH4, H2 data because it does not direct analyze and not agree with CH4 concentration.

AUTHORS: According to comments, we removed data related to CH4 and H2 from Table 3. At the same time, we removed the stoichiometric calculations of methane and hydrogen as well as the corresponding reference from the text. (Wolin, M.J. A Theoretical Rumen Fermentation Balance. J. Dairy Sci. 1960, 43, 1452–1459.

REVIEWER 3: L339 not cleared related to Table3.

AUTHORS: The sentence was removed from the manuscript.

 

Author Response File: Author Response.pdf