Altering Methane Emission, Fatty Acid Composition, and Microbial Profile during In Vitro Ruminant Fermentation by Manipulating Dietary Fatty Acid Ratios

Round 1

Reviewer 1 Report

Introduction

L29:  Conclusion should not focus only on the positive effect. It should state the overall effect. Conclusion should also include that HN6 can reduce TVFA concentrations and pH.

 

L41-43: “. For example, dietary supplementation with octadeca carbon FA (C18-FAs) seems to reduce methane emissions from ruminants due to 42 biohydrogenation and the toxic effects of free FAs on both methanogens and protozoa [6].” Why is this sentence here when the previous sentence was related to N6 and N3 FA on gene expression, etc? Make coherent in writing.

 

L44: , “biohydrogenation inhibits PUFA absorption”. Is there any evidence for that, I do not think so, rather it will be opposite.

 

L45-47: “However, some studies have reported that despite biohydrogenation of unsaturated FAs, feeding cows higher amounts of n-6 and n-3 PUFAs altered meat FA composition, cellular function, and animal performance [7,8].” I could not find the logic of this sentence. It is usual that biohydrogenation of PUFA affects meat and milk fatty acid composition, etc.

 L58: “and are out of date” – I cannot understand why you said “out of date”. I will say those studies are highly important.

 L58-60: “Currently, the effects of fats from extruded oilseeds on in vitro rumen fermentation and microorganism-associated changes in methane production have not been explored.” I do not think so. There are many studies even in vivo. Moreover, why is this statement here? Was extruded oil sources were of you research focus?

 

Authors should justify it properly. There are many studies on fat for methane emission and ruminal fermentation. I really do not find any good explanation for this in vitro study. Introduction needs improvements. Authors should also present ingredient and nutrient composition of diet in table as composition could affect fermentation.

 

Methods

Authors studied the effect of N6 to N3 FA ratio on ruminal fermentation and methane production, but they did not analyse the ratio in the feed. Authors must analyse the fatty acids in feeds, if the intended ratios were maintained in the diet and provide detained FA composition in feeds. Was PUFA content similar among the diet?

 

L106-118: was the anaerobic condition maintained? How was it maintained? I did not find any information on it. Ruminal environment should be O2-free.

 

Results:

Table 1: P-value for treatment for pH, MCP and acetate was >0.05, but authors have present superscript letters to show treatment effects and stated “Different superscripts indicate significant differences in the indicators among groups (P < 0.05)”. So, this is contradiction.

 

Figure 2: is the unit for Figure C correct? Only 0.10 to 0.2 ml/h for maximum gas production?

 

Figure 3a: methane composition is not useful, but present methane production, i.e., ml/g DM.

Figure 3c: It seems the fermentation was not made properly. The rumen microbes are very sensitive to O2. In this O2 concentration, many microbes will die. What was the reason that MN6 increased O2 concentration? I do not think it is treatment effect.

 

Discussion

L413-415: “In particular, studies have reported that supplementation with C18 PUFA-rich oils inhibited methane production in vitro [68] or in vivo [69], and the extent differed depending on the degree of unsaturation and inclusion level [6].” Replace the reference without other study. Did these study evaluated methane production. After checking the references, it seems not. Why do you specifically focus on C18 PUFA in discussion? Your study was not on C18 PUFA, but N6 to N3 ratio.

 

I have seen authors have given a general discussion, not specific to the results obtained in this study. Please explain the following

1. Why ammonia concentration was higher for MN6?

2. Why MCP was lower for MN6?

3. Digestibility was similar, but VFA was lower for HN6. Why?

4. Why was O2 concentration lower for LN6 than for MN6?

5. What was reasons for interaction effect for TVFA and other FA?

6. What were the reasons that C12:0, C14:0 increased, C16:0 decreased with time? What was the reason for changes in C22:0 with time? I have doubt on these results.

Check all references if they were cited in the context properly.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

This study evaluated the effects of different dietary n-6/n-3 polyunsaturated fatty acid (PUFA) ratios on in vitro ruminant fermentation. The work is well written and very clear. The results and discussions are exhaustive. Moderate English changes required

Author Response

Dear reviewer and editor,

We thank the reviewer for your detailed feedback and helpful advice. In the revised manuscript, we have carefully addressed the comments provided by the reviewers. Additionally, a native English-speaking editor helped improve the writing quality of the text. Detailed point-by-point responses to the reviewer comments are listed below and we have included the new content or cited line numbers in the updated manuscript. All the relevant changes in the revised manuscript have been marked in red font for ease of review. Once again, we thank the reviewers for your comments, which have provided us an opportunity to improve our paper.

Best regards,

Xiaoge

 

Reviewer 3 Report

Some paragraphs need to be clarified in detail.  In the text, I indicate some suggestions in this regard

Comments for author File: Comments.docx

Author Response

Dear reviewer and editor,

We thank the reviewer for your detailed feedback and helpful advice. In the revised manuscript, we have carefully addressed the comments provided by the reviewers. Additionally, a native English-speaking editor helped improve the writing quality of the text. Detailed point-by-point responses to the reviewer comments are listed below and we have included the new content or cited line numbers in the updated manuscript. All the relevant changes in the revised manuscript have been marked in red font for ease of review. Once again, we thank the reviewers for your comments, which have provided us an opportunity to improve our paper.

Best regards,

Xiaoge

Round 2

Reviewer 1 Report

Related to my previous comments:

1) Response 8: Authors studied the effect of N6 to N3 FA ratio on ruminal fermentation and methane production, but they did not analyse the ratio in the feed. Authors must analyse the fatty acids in feeds, if the intended ratios were maintained in the diet and provide detained FA composition in feeds. Was PUFA content similar among the diet?

Response 8: Thanks for your question. The n-3 concentration, n-6 concentration, the ratio of n-6/n-3, unsaturated FAs, and EE, were analyzed. The detailed information has been shown in Table S1.

Comments: I feel authors should present the FA composition with the main manuscript not as supplementary file because it is important in the study context. 

Also FA analysis in feed s needs to be mentioned in the M&M.

2) Response 10: You should clearly state in the statistical analysis when you used Tukey test. When ANOVA is P>0.05, you cannot say, treatments were significantly different.

3) Response 13: You should clearly state the limitation of the study that it contained high amount of oxygen that might alter fermentation process. Otherwise other researchers will point out your paper and the science will be manipulated.

4) Response 17: Your in vitro study was not conducted well and thus you got high O2. You probably did not  follow proper anaerobic process. Did you use indicator to suggest that the media was anaerobic?

5) Response 19: Please provide citations that "the fatty acids < 16 carbon mainly come from de novo synthesis using VFA. Therefore, C12:0 and C14:0 increased with time" for the ruminal environment. 

Author Response

Please see the attachment

Author Response File: Author Response.docx