The Action Potential of Antioxidant Grape Seed Proanthocyanidin as a Rumen Modifier to Mitigate Rumen Methanogenesis In Vitro

Round 1

Reviewer 1 Report

This is an interesting manuscript about using using a natural product, grape seed proanthocyanidin, as a feed additive to decrease the amount of methane produced by cattle. There are some changes and modifications suggested for the authors to consider.

Abstract

Line 16; change to: 'To explore its potential as a rumen modifier'

Line 20; Change 'Increasing' to 'increasing'

Line 21; something appears to be missing here '(RmaxS,  /h)'

The authors indicate several times in the abstract that a difference is statistically significant (Lines 22-23, Line 25, Line 26, Line 29) but in each case should also indicate which tests were used to make these determinations.

Line 29; the '(P<0.05)' should be moved to Line 27 following the text that says 'the GSP addition significantly decreased the ratios of methanogens to total bacteria'. This is the only place in the sentence that refers to a difference.

Line 28; change to ' methanogenic archaea was not affected in either the HY or the LY group'

Line 29; change to 'that the rumen archael community'

Line 31; change 'under' to 'in'

Line 32; change 'methanogens' to 'methanogen'

Lines 32-33; change to 'In brief,'

Line 35; change 'under' to 'in'

Introduction

Line 44; change to 'there has been increasing interest'

Line 47; change 'was' to 'has been'. Also add '(GSP)' after 'grape seed proanthocyanidin'

Line 48; remove 'the grape seed proanthocyanidin' from the sentence and just use 'GSP'

Line 60; change to 'based on its action'

Line 63; change 'farm' to 'farms'

Lines 66-67; change to 'determine the supplementing'

Line 68; change 'diets' to 'diet'

Line 71; change to 'the abundance and diversity of rumen archaea.'

Materials and Methods

The authors could replace 'grape seed proanthocyanidin' with GSP because the latter has been introduced previously.

Line 84; the authors refer to Table 1, however, in my downloaded copy there is no Table 1 present.

Line 87; change to 'and were housed'

Line 89; change 'TMRs' to 'TMR'

Line 93-94; the authors need to add a reference for readers to be able to obtain the 'Guidelines of the Beijing Municipal Council on Animal Care.

Line 98; change to 'GSP doses' and 'based on a previous'

Line 99; change to '120ml served'

Lines 112-113; change to 'plotting a scatter diagram which provided a visual range for the user'

In the paragraph from line 114 to line 133, the authors use the word aliquot several times. Aliquot implies that the sample was divided into a number of equal volumes. This is not the case as the volume of the 'aliquots' in the paragraph are listed as different. Instead of the word 'aliquots', the authors should replace it with the word 'subsample'.

Line 116; change 'were' to 'was'

Line 131; change to 'gas sample in the air-bags was'

Lines 134 and 136; replace '16S rDNA genes' with '16S rRNA gene'

In the paragraph (Lines 143-156) the authors describe 2 sets of primers for doing the nested PCR for archaea. Did the authors design these primers? If so, they need to state that they did so. If they did not, they need to clearly indicate where they found them (i.e., references).

Line 144; change 'Firstly' to 'First'

Line 149; change 'descried' to 'described'

Line 164; the authors should provide a reference for Mothur

Section 2.7 Quantitative realtime PCR analysis

Overall, it is important to realize both for the authors and for the readers that this is not measuring the total number of cells. It is estimating the number of gene copies and this needs to be clearly indicated here and throughout the manuscript. So any ratio determined from these measurements does not reflect differences in cell numbers, but gene copy numbers, and different species can have a varying number of gene copies. 

Line 179; change 'determined' to 'estimated'

Have the PCR primers here been developed by the authors? If so, they should state this in the text. If not, they need to clearly indicate where they found them.

Lines 187-188; should read 'Amplifications were'

One critical analysis that is missing here is the running of melt curves to show that the primers only amplified one product. If the authors performed these analyses then they need to state this and indicate the results for the readers.

Line 197; should read 'incubation times'

Results

Line 307; should read 'ratio of methanogens'

Discussion

Line 359; change 'have' to 'has'

Line 372; change 'abundances' to 'abundance'. Remember to note that this was determined by measuring gene copy numbers.

Lines 406-407; should read 'incorporation'

Line 414; should read 'methanogen'

Lines 427-429; It is important to remember that if data cannot be shown to be statistically different then there is no difference even if they appear to be 'numerically lower'. This should be rewritten or eliminated from the text. 

 

Changes have been suggested in the comments to the authors.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors, the paper brings a work that seems to be well-conducted, however, I believe that analyzing effects of proanthocyanidins (known as condensed tannins) on ruminal in vitro fermentation seems to be a topic that lacks novelty, which becomes quite clear when the authors mentioned several literature data that support their findings. Considering that the paper deals with only in vitro asays, I would recommend a review, aiming at providing a different approach on data analysis, with a stronger focus on the results obtained in their itself than just comparing it to literature data. The microbial work done is interesting, and considering that very significant amount of information on proanthocyanidins is available on the literature, I believe that authors could make inferences about the structural properties of GSP, its potential for being used in ruminant nutrition and etc.

Please see below some specific aspects that can be improved.

 

Abstract

Line 17 – mg per incubation is a very vague information.

Keywords – I would not use words that are already present in the title as a keyword.

 

Introduction

Line 46 – you have used the chemical symbol for methane (CH₄) here. please use it in the rest of the text as well, where suitable.

Line 50 – “…has attracted attention”

Line 59 – 60 – It sounds confusing, please rewrite.

Line 61-63 – provide a reference for the info presented here

Material and methods

Please provide the name and brands for the manufacturers of the gas production system used here and its accessories as well.

Line 88 -89 – please provide diet ingredients as a proportion and not only as a wight measuring unit.

Line 97 – presenting the amount of GSP included as g per incubation fluid is extremely inaccurate. Suggest presenting it as a proportion of substrate (e.g. gram GSP per gram of diet);

Line 134 – You do not need to mention results here.

Results

Line 234 – what is the meaning of HF and LF?

Line 258 – replace has with had

Line 264 – Did you mean “non glucogenic?”

Line 328 – The text continues here?

 Discussion

The discussion is short and authors dedicated a significant part of it to present literature results that are somewhat quite common or expected (e.g. plant secondary compounds that reduced gas and CH₄ production or feed digestibility). I believe it should be written again providing a more in-depth discussion of data obtained in their assay. More focus on the explanation to different results between HY and LY substrates should be given.

Line 365 – 367 – Repeating what is already described in the methods

Conclusion

it is not wrong, but it is very expected that proanthocyanidins would cause this effect in rumen.

 

 

Figure 1 – Improve figure quality, very hard to distinguish between lines.

An English review would be beneficial  to the paper. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The paper seems very interesting, but the authors have  to resend it in a complete form which include table 1 which I could not find as well as the numeric results concerning IVDMD. 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf