Review Reports - Combining Crude Glycerin with Chitosan Can Manipulate In Vitro Ruminal Efficiency and Inhibit Methane Synthesis

Round 1

Reviewer 1 Report

The manuscript reports the evaluation of glycerol and chitosan inclusion in a TMR diet on CH4 production, rumen fermentation and digestibility. At the beginning I found the study methodologically sound, however I have a few major concerns that to my opinion need to be addressed prior being suitable for publication.

Major Concerns:

1.- My main concern is that VFA’s and CH4 were measured just at 2 and 4h of incubation, and not at the end of the fermentation. So, their conclusions might be erroneous as CH4 concentrations/production could have been equalized throughout the fermentation process: This must be at least be stated in the discussion. A refer that CH4 is reduced in the early fermentation.

2.- For CH4, NH3-N, pH, VFA’s and digestibilities authors have 2 incubation times, which to my opinion must be incorporated in the statistical model as a repeated measure

3.- When having a look at GP data with negatives values for a, it suggests that authors should have used a model with a lag phase. I think authors need to model the data, including that lag phase

4.- Gas production and CH4 data must be reported on a 1g basis, in order to be more comparable with other studies

5.- For all tables, the Glycerin*chitosan interaction was reported, however none of the evaluated variables was significant. Thus I recommend to report all tables with means of main effects separately and the p-values and sem for glycerin and chitosan and p-value for the interaction, and state in the results section that the interactions were not significant.

6.- I feel that the language must be improved, I tried to help with some changes, but think it needs a deeper revision

Minor comments

L19: Please define C3 at first mention

L21: replace “were” by “was” and delete “to”

L22: add “arrangement of treatments”

L32: mg% is the unit correct?

L40: acid”s”

L41: propoionate (C3) should have been defined in line 19, and here you can use C3

L45: Do not start a sentence with an abbreviation, please change C3 for propionate

L53: please delete CH4, it was already defined

L54: Do not start with an abbreviation

L58: please delete C3, it was already defined

L58-59: This sentence is a little bit unclear, please rephrase. I would say something like that “propionate serves as an H sink in the rumen, thus an increase in its concentration would reduce H availability and therefore CH4 production”.

L62: Please first mention that van Cleef et al indicated the possibility of reducing CH4, and the start mentioning the negative effects.

M&M

The ingredients of the TMR and their proportions (control diet) must be described

L89: Delete “in”

L90: delete “be”

L93: I would say residue instead of remainder

L104: as rumen fluid donnors

L107-108: ……. was mixed at equal proportions.

L113: replace “were” by “was” and delete “to”

L114-115: treatments are the combination of the 3 levels of glycerin and 3 levels of chitosan.

L124: A digestibility evaluation was arranged ……..

L122: Replace “The” by “A set of”

L127: Chemical analyses instead of assay

L132-133: Check spelling. I recommend “…. was analyzed for CP and OM with the similar protocol mentioned above”………

L138: was analyzed, instead of was investigated

L139: just use C2, C3 and C4

L142-145: How did authors correct for the microbial mass in the residue?

L145: Methane, do not start the sentence with an abbreviation

L150: Statistical analysis and gas kinetics

L151:was used for gas kinetics analysis.

L163: … was increased ……

L168-169: at what level of inclusion?

L175: which indicates ….. has a potential for animal supplementation use

L178: When having a look at GP data with negatives values for a, it suggests that authors should have used a model with a lag phase. I think authors need to model the data, including that lag phase. Besides, as the interaction was not significant, authors should report main effects instead of the interaction. GP data should be standardized on a 1 g/basis

L179: in terms

L193: The differences among trials should be discussed

L200: it is a buffered medium, so it is expected that pH levels are within those values. So please state that: Considering it is a buffered medium ………

L201: mg% unit for NH3-N, I think mg/100ml is better

L209: concentrations of CH4 could “be reduced”

L285: …… glycerin “incorporated” into TMR ……

L287-289: Please double check this sentence, How can be C3 at the expense of itself?

L292: van Cleef with uppercase (Van Cleef), since it is at the beginning of a sentence

L311: a mechanical “effect”?

Sincerely,

The reviewer

Author Response

Author's Reply to the Review Report (Reviewer 1)

Response: We appreciate thanks so much for comments and suggestion made on manuscript. Above all, the authors felt that all points made were very useful and have incorporated most of the corrections where necessary as suggested in order to make the manuscript ready for possible publication. All those corrected and modified appear in track change mode in the manuscript. Please see information given by the authors following the suggestions and comments made by all reviewers. Thanks!

Major Concerns:

Response: Thank you for the nice suggestion and now we have added into the text as “However, present study found CH4 was reduced in the early fermentation of 4 h of incubation, thus throughout the fermentation process (96 h of incubation) should be further elucidated.” Please see in section of “In vitro rumen parameters and CH4 concentration” in page 9.

2.- For CH4, NH3-N, pH, VFA’s and digestibilities authors have 2 incubation times, which to my opinion must be incorporated in the statistical model as a repeated measure

Response: We have re-calculated and provided detail in section of “Statistical analysis and gas kinetics” as “Data from 2 incubation times of CH4, NH3-N, pH, VFA’s (2 and 4 h of incubation) and digestibilities (12 and 24 h of incubation) were analyzed as a mixed model with repeated measurements using the PROC MIXED of SAS [17], in which the effect of sampling time was considered fixed and that of the inoculum was considered random. Data of in vitro gas kinetics and cumulative gas production were subjected to the General Linear Models (GLM) procedures of SAS [17]. Differences among treatment means for all parameters were contrasted by Tukey’s Multiple Comparison Test.” Please see in manuscript.

3.- When having a look at GP data with negatives values for a, it suggests that authors should have used a model with a lag phase. I think authors need to model the data, including that lag phase

Response: We have already re-analyzed regarding to your comments by choose the model of France et al. (2000; Br J Nutr, 83:143–150.) which including lag phase the following model:

Gas production = b × [1 – e^-c(t-L)]

4.- Gas production and CH4 data must be reported on a 1g basis, in order to be more comparable with other studies

Response: Thanks. Now, we have re-calculated for gas production and CH4 data and reported on a 1 g basis, in order to be more comparable with other studies. Please see in the tables and throughout manuscript.

Response: It is very great suggestion and we have all agreed with. Now, all Tables have been modified accordingly to your comment already. In addition, results and discussion was also revised wherever the Table changed. Please see in manuscript.

6.- I feel that the language must be improved, I tried to help with some changes, but think it needs a deeper revision

Response: Thanks so much for your comments and suggestion us to improving English. Now, we have revised accordingly your comments point-by-point as well as wherever it need to improve. Furthermore, before we submit manuscript to the journal, we have send our Manuscript to the company namely Papercheck (https://www.papercheck.com/) located in California, United States for English grammar edited already (Order no. 775639). Herein, we have attached receipt of payment for English grammar proof. Therefore, we think that throughout manuscript have been improved in English language.

Minor comments

L19: Please define C3 at first mention