Impact of Oil Sources on In Vitro Fermentation, Microbes, Greenhouse Gas, and Fatty Acid Profile in the Rumen

Round 1

Reviewer 1 Report

From the paper, it can be seen that the authors did a lot of research on acid production, microbial community and gas emission of rumen fermentation by different oils addition. Through which it was found that the addition of CO could increase the propionic acid production after 24h of in vitro incubation. Addition of CO and Ca salts effectively reduced the number of methanogenic archaea and addition of LSO effectively reduced the number of rumen ciliates. I think the paper could be published with the following shortcomings resolved.

1: The experimental data in this paper are from three studies with different additives, and there is no blank control group, so it is not known how effective the results of this study.

2: The value of the oils and products in this study was not assessed, is this of application significance.

Author Response

Thanks for your constructive comments. Your comments help us to improve this manuscript. The responses to comments are mentioned below:

From the paper, it can be seen that the authors did a lot of research on acid production, microbial community and gas emission of rumen fermentation by different oils addition. Through which it was found that the addition of CO could increase the propionic acid production after 24h of in vitro incubation. Addition of CO and Ca salts effectively reduced the number of methanogenic archaea and addition of LSO effectively reduced the number of rumen ciliates. I think the paper could be published with the following shortcomings resolved.

1: The experimental data in this paper are from three studies with different additives, and there is no blank control group, so it is not known how effective the results of this study.

Response: Thanks for your comments. Regarding the blank control, we just assigned the blank to confirm the proper ruminal incubation without the applications of FA as well as the synthetic diet. In some previous in vitro studies, these blanks, without the diet and additives, did not present in the tables (Weinberg et al., 2007 published at Journal of Dairy Science; Yuan et al., 2015 published at Animal Feed Science and Technology). However, your comment is so constructive for us, which the positive (with diet) and negative blank should be designed to confirm the main effects in our next study.

2: The value of the oils and products in this study was not assessed, is this of application significance.

Response: Clarified it.

Reviewer 2 Report

Authors investigated the impact of oil sources on in vitro fermentation, microbes, greenhouse gas, and fatty acid profile in rumen fluid. The manuscript is well written, and it reports interesting results since the reduction of greenhouse gasses in cattle combined with the optimization of nutrient utilization is a hot topic at this time. There are few sentences that need to be amended for the English language to be clearer for readers. I have a question for authors that maybe could be addressed also along the manuscript. Do you think that concentrations tested could be applied in animal nutrition or they have been only selected for in vitro test and they are not suitable for in field conditions? Do you think that Ca-salt fatty acids should be considered as the best solution to limit the methanogenic bacteria and impact less than possible the fibrinolytic classes of microorganisms?

I have few minor comments as listed below:

Abstract

Line 22-23: A brief description of the method should be provided.

Line 24-26: Each time that you refer to a statistically significant result the p-value should be listed.

Line 28-29: Highest compared to? When you refer to the highest/lowest the term of comparison should be provided.

Introduction

Line 49: Try to explain why the inclusion of pure FAs can be difficult.

Line 53: Revise the English.

Line 56: fatty acids.

Line 60: Please underline the fact that you refer to saponified fatty acids.

Materials and Methods

Line 78: Can you provide more information regarding animals’ diet? Which was the complete ingredients list of the diet? CP and NDF are for sure two important nutrients, but which was the concentrations of moisture, crude fibre, ADF, ADL, ether extract and ashes?

Line 81: Which amount of rumen fluid was collected? How much volume consisted of the final inoculum?

Line 83: Can you describe in brief the anaerobic culture medium?                                                                                                                                                                                                                       

Line 85: Correct with carbon dioxide. Which was the concentration of CO2?

Line 89: Which was the source of Ca-salt FAs?

Line 94: Clarify how did you prepare blanks.

Table 1: Did you analyse this composition?

Line 119: Which standard did you use?

Line 134: Specify that you evaluated the 260/280 ratio. Which cut-off did you consider as pure DNA?

Line 136: What do you mean for “general bacteria?

Line 138: Please provide the PCR conditions (amount of DNA template, final volume, concentration of primers).

Line 143: How did you prepare blanks?

Line 150: Did you include the random effect of the individual animal?

Results

When a result value is listed an error range should be provided. The Table 4 should be listed after the paragraph (lines 182-200).

Line 190: Please specify “compared to other groups”.

Line 191: Highest compared to which groups?

Line 194-195: Higher compared to which group?

Line 197: When referring to statistically significant results the p-value should be provided.

Line 198: Compare to?

Figure 1 and Figure 2: The overall p-value can be included for each figure of the panel. The significance level should with asterisks can be defined in the figure caption. It is not necessary to report all p-values and SEM since the figure represents means ± SEM and asterisks indicates the significance level. Which is the measure unit of Acetate and Propionate? Define in the figure caption how the results have been expressed (mg/g of what?).

Chapter 3.3: Please provide the measure unit to values (FC).

Line 226: Higher compared to?

Line 228-229: Compared to other experimental groups?

Figure 3-4: A similar approach that I suggested for Figure 1 and 2 could be also applied to this panel.

Line 267: Please always refer to a comparison also when listing the highest/lowest value.

Discussion

It is not necessary to cite again the Figure or Tables since they have already mentioned in the results section.

Line 309: Which could be the mechanism that caused this outcome?

Author Response

Thanks for your constructive comments.

Your comments help us to improve this manuscript.

Please find the attached response letter.

Thanks.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The quality of the manuscript has been improved. Authors have been replied to my comments and reviewed the paper according to most of my requirements. I have only few minor comments to clarify some aspects as listed below.

R2: Line 53: Avoid abbreviations and try to use a formal language for the English style.

R2: Line 83-84: Thank you for the clarification. In this sentence it should be distinctly stated that rumen fluids of two animals were bulked together. I suggest you revise “Approximately 1 L of rumen fluid was collected from both animals…”.

R2: Line 85: Thank you for your answer. My curiosity was mostly related to understand if you created an anaerobic atmosphere with 100% saturation of CO₂ or there was some oxygen or other gasses (nitrogen)?

R2: Table 1: If you analysed the composition of fatty acids from oil sources the method should be described, and these data could be listed into the results section. If these are results, an error range should also be listed.

R2: Line 161-163: How much DNA template did you add to the PCR reaction mixture?

Authors Response: The ratios of absorbance at 260 and 280 nm in our samples are ranged from 1.78 to 1.97. Therefore, we considered the 260/280 ratios with ≤1.78 are accepted as “pure” for our DNAs.

R2: This information should be added to the manuscript.

Authors Response: We have added the sentences, “, and the concentrations of all template DNA samples were adjusted to 50 ng/µl” and “PCR conditions were 95 °C for 5 min, followed by 35 cycles of 95 °C for 15 s and 56 °C~60 °C for 30 s”, and “200 nM”.

R2: These conditions should be added to the paper.

Authors Response: Error range did not list in the result value because the information of results provided in Table. Table 4 listed after the paragraph.

R2: The error range should be provided every time that you refer to a numerical value along the text.

R2: Do you think that concentrations tested could be applied in animal nutrition or they have been only selected for in vitro test and they are not suitable for in field conditions?

Authors Response: Clarified it.

R2: The inclusion of a statement to the discussion and/or conclusions chapter is recommended to clarify this point.

Author Response

Thanks for your comments.

Please find the attachment for our response.

Thanks.

Author Response File: Author Response.pdf