Effects of Dietary Capsaicin and Yucca schidigera Extracts as Feed Additives on Rumen Fermentation and Microflora of Beef Cattle Fed with a Moderate-Energy Diet

Round 1

Reviewer 1 Report

The paper is well designed and carried out correctly, but there are some aspects that deserve consideration.

There are some concepts that are wrongly presented, for example, enzymes are not excreted (L36) but secreted, and in any case some carbohydrates (fibrous polysaccharides) are not broken down by secreted enzymes, but by those linked to the bacterial surface. To my knowledge, the mention to Rubisco in Discussion (L341-344) is nonsense since this enzyme is related to plant photosynthesis. In Results, bacterial abundance is presented at the genus level, but results of some families are mixed up (i.e., L211-212). I am not sure that Bacteroidota is a valid name for the phylum, that I would say it is Bacteroidetes. I do not have any previous knowledge about Patescibacteria, but being a new phylum an indication of some known organisms now classified within should be useful.

The response to additives is based on their dose. The proportion of the tested additives must be clearly given (do 150 g/d capsaicin and 240 g/d yucca refer to the additive given or to the active compound? What was the concentration of the active compound in the extracts?) and justified (why was such dose chosen?). In this regard, I have a major concern: if diet (TMR) was offered ad libitum and was not measured (animals were housed in groups and intake was not recorded) how can be the doses assumed as per day basis? How can be ensured a fixed amount of additive ingested per day?  What was the proportion per kg of ingested feed?

It is difficult to see fermentation values in these figures, a table including errors and highlighting significant differences by letters should be clearer. Some fermentation results, mainly TVFA, are unexpectedly low for treatments CON and CAP, and differences with YSE are too large. Can this be caused by the sampling procedure (stomach tube)? Anyway, this must be discussed and justified. Also regarding fermentation, no differences were recorded on propionate proportion, but the effect of YSE is justified according to this parameter in L329-331.

According to the experimental design, rumen contents are sampled once at a fixed time (before the morning feeding), so it gives a fixed picture of what occurs then and do not show trends of fermentation pattern. Therefore, some statements proposed in Discussion section related to the pattern of fermentation (like pH, L277-280 and L285-287) are out of the scope of the paper. Further, lactic acid (L280-284) or methane (L320-325) were not measured here, so comments about them are not related with the work done. Too general statements not directly related with the work done (i.e., L291-298) should be avoided. In other way, results like the presence of protozoa, which are basic for understanding rumen fermentation and are greatly affected by saponins, are mentioned in the Introduction section but not measured, thus giving a partial picture of rumen microbiota.

Table 2 gives up to 4 different diversity indexes, all of them showing the same ranking of treatments. Are all necessary? In any case, an explanation of what do they indicate, and a small discussion of their results should be valuable.

Are all graphs necessary in Figure 3? 3b and 3c do not match with the figure title. I cannot understand Fig 3f.

In Discussion, authors assume that an effect of YSE on bacteria may justify a response in fermentation (differences between CON and YSE), but similar effects of Cap on microbiota were not reflected on fermentation parameters respect to those with CON. This must be highlighted and explained. In L379-386, I cannot understand the linkage among statements justifying the reasoning.

Conclusions are too general and do not specifically linked to the results obtained. Besides, they are extended to bot additives, whereas those from CAP differ to those from YSE.

In terms of style, English needs to be revised. Do not start a sentence with “But” (L20) or “And” (L48). In order to avoid confusion, do not the same abbreviations for the products and for the treatments (CAP and YSE). There are some repetitions that need to be rewritten, like “plant extracts are bioactive substances extracted…” (L43), “inhibit cellulolytic bacteria and fungi and inhibit cellulolytic bacteria and fungi” (L58-59) or “the dominant bacteria at the level of ruminant bacteria” (L310-311).

Author Response

Dear Reviewer，

Thank you for your letter dated December 5. We were pleased to know that our work was rated as potentially acceptable for publication in Fermentation, subject to adequate revision. We thank the reviewer for the time and effort that you have put into reviewing the previous version of the manuscript. Their suggestions have enabled us to improve our work.  Appended to this letter is  our point-by-point response to the comments raised by the reviewer. The comments are reproduced and our responses are given directly afterward in a different color (red).We would like also to thank you for allowing us to resubmit a revised copy of the manuscript. Please see the attachment.

We hope that the revised manuscript is accepted for publication in the Fermentation.

Sincerely,

Xin Yi

Author Response File: Author Response.pdf

Reviewer 2 Report

General

All opinion was emitted with all respect to the author's effort for the preparation of the experiment and its report

The purpose of this study was to evaluate the effects of dietary capsaicin and yucca schidigera extracts used as feed additives on rumen fermentation and rumen microflora of beef cattle that were fed with moderate-energy diet (characterized as growing diet for feedlot system). In general, the topic of the study is pertinent. The antecedents and justify of the study are well described and supported. In addition, the manuscript is supported with excellent methods, techniques and procedures. The experimental design and statistical analyses are appropriate. However, the manuscript has flaws that limit it consideration for publication in Fermentation journal and be listed below

Title: Needs be more descriptive

This type of feed additives acts differently depending on the type of diet. The magnitude of their effects is not the same on cattle fed a growing diet (containing moderated soluble carbohydrates and high NDF level) than on cattle that are fed with finishing diets (containing high soluble carbohydrates and low NDF level). Therefore, the type of diet must be mentioned in title (hint) “Effects of dietary capsaicin and yucca schidigera extracts used as feed additives on rumen fermentation and rumen microflora of beef cattle that were fed with moderate-energy diet”

Abstract

L12: remove similar body weight (subjective). Please mention de average weigh (kg) and its SD (hint): 45 healthy angus (initial weight=XX ±XX kg)

Introduction-Justification: Must be improved

The central justification is that there exists a lot of information about the effects of saponins and capsaicin on rumen fermentation in vitro, but the validation results of in vivo are lacking. This is not true, several reports about the effects of saponins and capsaicin on ruminal fermentation in vivo are available, to name a few: Wu et al. 1994. Journal of Animal Science 72(4):1038-42; Zinn et al. 1998. J. Anim. Sci. 76 2239–2246; Liu and Li (2011). Advanced Materials Research 343-344:655-660; Wang et al., 2019. For capsaicin (Rodriguez-Prado et al. 2012. J Anim. Sci.90(6):1879-84; Alford et al., 2016. J. Anim. Sci. 94 (Suppl 5)801.

A proper justification is that the inconsistencies about the effects on ruminal fermentation and microbial population of these extracts evaluated in vivo in previous studies could be resulted by type of diets and by duration of period evaluation (short periods <15 d). To our knowledge, no evaluations of the effects of these extracts on ruminal fermentation and microbial population on long term periods are available. Our hypothesis that capsaicin and yucca extracts could change the rumen bacterial community structure, thereby affecting rumen fermentation even when supplemented at long-period to cattle fed a moderate-energy diet. For this reason, the purpose of this experiment was...

 

Results and Discussions. Must be improved

It is well known that VFA patterns (total and molar proportion) are strongly affected by dry matter intake level (Noziere et al., 2011). For that, it is essential that DM intake data of each treatment must be included in Tables. If there were differences between treatments on DMI, this must be considered as a factor that can affect the results on ruminal fermentation and microbiome and must be discussed. 

Specific

Material and methods

L69: remove similar body weight (subjective). Please mention de average weigh (kg) and its SD (hint): 45 healthy angus (initial weight=XX ±XX kg)

L70: Use “animals” instead “heads” to describe the experimental units

L86: Please rewrite as: to forages ratio to meet nutrient levels for growing cattle recommended by NASEM [17]. In addition, include NASEM in references!

L87: Please rewrite as: Diet formulation and nutrient composition (DM basis) of basal diets are shown in Table 1

L99: Why weighed the cattle and this information is not shown in Tables and not discussed? Include information of initial and final body weight, the average daily gain, and feed efficiency, or remove the weight procedure from mat & methods (only mention that cattle were weighed upon initiation of the experiment)

Table 1 L95: is metabolizable energy, not “metabolic’. In addition, indicate from what source ME was calculated (i.e., NASEM? ARC?) specify please

Discussions. Must be improved

It is well known that VFA patterns (total and molar proportion) are strongly affected by dry matter intake level (Noziere et al., 2011). For that, it is essential that DM intake data of each treatment must be included in Tables. If there were differences between treatments on DMI, this must be considered as a factor that can affect the results on ruminal fermentation and microbiome and must be discussed. Discussion about of the effects (or absence of the effects) of capsaicin on rumen fermentation and bacteria are absent. Please, contrast your findings with previous reports and argue

Specific

L265-269: This statement indicates that previous studies had demonstrated that YS has a NH3-N inhibitor effect. And this confirms your results, but, your results show the opposite! YS increased NH3-N. Please clarify!

L287: VFA produced in the rumen of beef cattle in a day and night?? Please rewrite as:  VFA produced in the rumen can account for 50~75% of the energy required by the body.

L303: "producing greater numbers of microorganisms in the rumen, reducing the microbial protein to be synthesized by the rumen" This statement is illogical! Please clarify.

L337-338: Here and through the discussion, it is imperative that mention the specie, type of diet (forage:concentrate ratio) and type, source, and the dose of extract used (if that's the case) in the cited reports  that are used to sustain your findings. . In order to not confuse the readers. For example, the references 48-49 are results obtained in mice! And this must be mentioned

Conclusions

Be more specific in your conclusions. In this experiment CAP y YS improves some parameters of rumen fermentation but impairs others.

In fact, the extracts had different effects on ruminal fermentation. YS increase TVFA (good), but no affect AC:PR ratio. YS increased NH3-N and lowered ruminal pH (not so good)

CAP decreased NH3-N (good), decreased TVFA (not good), without effects on other fermentation patterns.

Both, CAP and YS increased bacteroides and decreased firmicutes.

Please expose your conclusion taking this highlights

Author Response

Dear Reviewer，

Thank you for your letter dated December 5. We were pleased to know that our work was rated as potentially acceptable for publication in Fermentation, subject to adequate revision. We thank the reviewer for the time and effort that you have put into reviewing the previous version of the manuscript. Their suggestions have enabled us to improve our work.  Appended to this letter is  our point-by-point response to the comments raised by the reviewer. The comments are reproduced and our responses are given directly afterward in a different color (red).We would like also to thank you for allowing us to resubmit a revised copy of the manuscript. Please see the attachment.

We hope that the revised manuscript is accepted for publication in the Fermentation.

Sincerely,

Xin Yi

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I have read the corrected version of the manuscript and appreciate the authors' consideration in satisfactorily attending my suggestions made to the original version of the manuscript. I have no further reviewer comments. In my opinion, the manuscript is now ready to considered for publication.