Milk Fatty Acids: The Impact of Grazing Diverse Pasture and the Potential to Predict Rumen-Derived Methane

Round 1

Reviewer 1 Report

I have reviewed the manuscript entitled "Milk Fatty acids: the impact of grazing diverse pasture and the potential to predict rumen derived methane" (identified as agriculture-2059546). It is a very comprehensive, laborious, and long-term study. The study's topic and objectives are within the scope of the journal. A sufficient amount of information is provided in the introduction, and the researchers have clearly stated their objectives. In other sections, there are, however, a few major shortcoming  that need to be really fixed. There is, however, a possibility that it may be one of the good works that will attract attention in its field of expertise if the following conditions are met.

Major comments:

It is recommended that you follow the journal's guidelines when writing the heading of subsection.

It appears that the abstract was drafted independently of the article text, so it is recommended to harmonize it with the main text.

L121-127: Why are the corresponding values for the diverse sward equal to 99.5% and not 100%?

In several places in the text, “simple sward” and “grass clover” are used interchangeably. This is also true of the terms "house cows" -"silage and “diverse sward grazing”-“herbs”.  It is quite confusing to use them differently in the text, tables, and graphics, which negatively affects the flow of the text.

In "Materials and Methods" section; could you please clarify how many milkings per day were made, and were they mixed proportionally to their volumes in order to obtain a representative sample?

It would be helpful if all Pearson correlation statistics could be presented in a table or in the form of a correlagram (Table 2a and Table 2b).

A clear statement of equations 1, 6, 15 and 17 should be included in the material and method section. L202-209 contains values obtained by applying these equations. These values, as well as means and standard error of means of the recorded values should be presented in tabular form, and the text should refer to the table. In addition, the text should only contain means, whereas the table should contain means and SEMs.

L178: It is recommended that there is a space between the paragraph and the table title. Superscript should be used for lettering and certain sections of the table be separated by a line. Please use “p-values” instead of “ANOVA p-values*”. Please use “d9 activity ” instead of “d9 activity % (C14)”. Furthermore, please remove the word”key” and “fatty acid abbreviations” from footnotes and use "Monounsaturated fatty acids" and "Polyunsaturated fatty acids" instead of "Monounsaturated acids" and "Polyunsaturated acids". Please use “(silage, simple sward grazing and diverse sward grazing)” instead of “(Silage, Simple sward grazing and Diverse sward grazing)”. Please use “Fatty acid groups” instead of “Fatty acid groups and ratios”.

Could you please explain how the correlation results in Table 2a were derived? "C20:0", "C20:4 n6", "CLA3 (t,t)", "CLA2 (t,t)", "t11c15 C18:2", "ct mix 10,14/12 , C18:1", "c14+t16 C18:1" values are not given in Table 1. Table 2b presents a similar situation.

In the entire text, please use either "ECM" or "EC milk".

L236-238: It has been stated that 24 spring block calving organic Jersey cows have been used in this study. However, you noted here that the equations are used to plot the 40 estimated values against the recorded methane emissions. Can you explain this difference? If possible, please provide more details regarding how you achieved these results in the material method section.

The unit of fatty acids is "g/100g" in L145, but it is "%" in the following places and tables. Please correct this inconsistency.

It would be helpful if you could review L473-478 and remove any that do not pertain to you.

Minor comments:

L6-14: It is necessary to rearrange affiliations in accordance with journal writing guidelines.

L55-56: Please use “…. quantity; however, …” instead of “… quantity however….”.

L64-65: Please use “associated GHG and financial cost” instead of “and associated GHG and financial cost”.

L77: Please remove “(FA)”.

L97, L108, 174, 432: Plase use “Loza et al. “ instead of “Loza, Reinsch, Loges, Taube, Gere, Kluß, Hasler and Malisch”.

L133: Since “SCFA” is not used in other parts of the text, it must be clearly stated.

L134: Please use “… Stergiadis et al. [24]” instead of “… Stergiadis, et al. [24]”.

L135: Please use “gas chromatograph” instead of “Gas Chromatograph”.

L137, 139, 140, 141, 142, 143: Please give a space between “…” and “°C”.

L150-170, L442-450: Line spacing should be checked.

L151: Please use “Tukey’s Honestly Significant Difference” instead of “Tukey Honest Significant Difference”.

L156-157, 203, 365, 378, 385, 407: “Bougouin et al. [21]” instead of “Bougouin, Appuhamy, Ferlay, Kebreab, Martin, Moate, Benchaar, Lund and Eugène [21]”.

L175: Please use “EC milk yield” instead of “ ECM yield”.

L186: In relation to CLA9, please correct the mismatch in Table 1 and L186.

L193: Please use “n6:n3 and SFA:PUFA” instead of “ ratios of n6:n3 and SFA:PUFA”.

L195-200: If you are sorting i, ii, iii, and iv, do not continue as 5th.

L260: Please use “Soder et al. [28] reported ” instead of “Soder, et al. [28] do report”.

L270: Please use “Palladino et al. [29]” instead of “Palladino, et al. [29]”.

L276: Please use “Palladino et al. [29]” instead of “Palladino, Buckley, Prendiville, Murphy, Callan and Kenny [29]”

L106, 110, 118, 176, 177,  Table 2a footnote, Table 2b footnote, 261, 277: “/cow/d” instead of “per cow per day”.

L279: Please give a space between “8-9” and “kg”.

L284, 288, 292: Please use “Butler et al. [31]” instead of  “Butler, Stergiadis, Seal, Eyre and Leifert [31]”.

L292: Please use “VA ” instead of “vaccenic acid 292 (VA, t11 C18:1)”.

L294: Please use “CLA9 ” instead of “conjugated linoleic acid (CLA9, c9t11 C18:2)”.

L300-302: Please provide references.

L306, 325: Please use “Stergiadis et al. [13] ” instead of “Stergiadis, Hynes, Thomson, Kliem, Berlitz, Günal and Yan [13]”.

L309-310, 326: Please use “Leiber et al. [14] ” instead of “Leiber, Kreuzer, Nigg, Wettstein and Scheeder [14]”.

312-313, 327: Please use “Mangwe et al. [15] ” instead of “Mangwe, Bryant and Gregorini [15]”

L314: Please use “Butler et al. [33]” instead of “Butler, et al. [33]”.

L328-329: Please use “Butler et al. [33]” instead of “Butler, Collomb, Rehberger, Sanderson, Eyre and Leifert [33]”.

L382: Please use “Pearson’s corelation coefficient” instead of “Pearson’s coefficient”.

L401-402: Please remove parentheses.

L418: Please use “Poteko et al. [35]” instead of “Poteko, et al. [35]”.

L428: Please give a space between “67” and “g”. Please use “kg”  and “and” instead of “Kg” and “&”.

L434: Please give a space between “253” and “9” and “g”.

L434, 435: Please use “methane” instead of “CH4”.

L436: Please use “Poteko et al. [35]” instead of “Poteko, Schrade, Zeyer, Mohn, Zaehner, Zeitz, Kreuzer and Schwarm [35]”.

L447: Please use “FA” instead of “fatty acid”.

 

Author Response

Thank you. for the helpful feedback on our paper.  Our responses are in the attached document which I hope addresses all the points you make

Author Response File: Author Response.pdf

Reviewer 2 Report

In this article, the authors conducted an evaluation of parameters such as fatty acids in milk and methane production when cows grazed different types of pastures. Among their primary findings were that cows produced milk with higher levels of beneficial fatty acids compared to that from ryegrass/white clover swards, which in turn was superior to milk from the mixed silage diet. After this they make a couple of relationships between milk fatty acid profiles and methane emissions and then replace their data in other previously published equations, however, the paper has some shortcomings mainly in methodology and results. Some of these are described below:  

Lack of including information about the experiment in the methodology, e.g., age of forages, methane measurement, chemical composition of the forage-diets, equipment used, 

Failure to include the numerical p-value and mean square error of the model in the tables of results. 

The description of the statistical model used is missing. 

The comparison made between predicted and actual values is incorrect, a deeper analysis should be performed, for example, the following should be clarified: average, standard deviation, mean, concordance correlation coefficients (CCC), mean squared prediction error (MSPE), errors of central tendency (ECT), errors due to regression (ER) and errors due to disturbances (ED), among others.

Results such as dry matter and nutrient consumption and their relationship with fatty acids and methane have yet to be shown.

Author Response

Thanks you for feedback on our paper - I hope the revised paper on the basis of comments from other reviewers make our paper more acceptable

Author Response File: Author Response.pdf

Reviewer 3 Report

Manuscript ID: agriculture-2059546Milk:  Fatty acids: the impact of grazing diverse pasture and the potential to predict rumen derived methane.

The manuscript aimed to evaluate the effect of diverse forage pastures on the nutritional quality of milk and methane emissions from Jersey cows.

Although not new, this manuscript presents interesting information that can contribute to the study of the effect of forage type on milk composition and methane production by dairy cattle. However, the authors need to work hard before their manuscript is ready for publication in Agriculture.

Abstract

I do not think the abstract presents enough evidence to reinforce that milk fat composition can be a tool to predict CH₄ production.

Introduction

Line 37: please rephrase this sentence: "at least ticking as many boxes as possible" It does not read well, and its meaning is vague.

Line 44: Add a reference after "inefficient use of human edible food" and argue why cattle production is an inefficient form of human food.

Line 45: Mention the sustainability targets.

Line 50: This form of intensification also increases the carbon footprint because of the emissions associated with the production of crops. You may want to add a sentence to complete your idea and the corresponding reference.

Line 53: Why does enhancing the nutritional quality of milk results in improved sustainability of milk production systems?

Line 60: I believe the n6:n3 ratio is wrong. More n6 is needed for better cardiovascular health. The ideal is ~6.1. You may want to check:

DIETARY n-6 AND n-3 FATTY ACID BALANCE AND CARDIOVASCULAR HEALTH

AU  - Wijendran, Vasuki. AU  - Hayes, K.C. Y1  - 2004/06/09. PY  - 2004. DA  -

doi: 10.1146/annurev.nutr.24.012003.132106

Line Line 74: Add "diet" after changes.

Line 88: feeding management?

Materials and methods

Indicate the experiment's total duration and the number of experimental periods.

Line What was the duration of the rumen washout periods between sward types? Also, it is unclear if the three forage types were in the two 2.2-ha paddocks.

Line 123: It is unclear here if cows grazed on the three forage types mentioned in line 100.

Line  127: simple? Or sample?

Line 150: Continue using double-spaced text as in previous paragraphs.

Line 158: Use “model” instead of equation throughout your manuscript.

I find it difficult to follow the different ways the authors use to refer to the three treatments. They must use one term for each treatment and use it consistently.

Results

Table 1: Use throughout the manuscript the same description to refer to the three treatments. E.g. In line 100 a different treatment name is used, which differs from the names in Table 1.

Line 174:For more than two authors use “et al.”

Line 185: Housed cows is a new treatment?

Line 236: Need to mention how many time CH4 was measured in the 24 cows?

Line Line 248: Add “that” after evidence.

Present your results for methane emissions by individual treatment. Also include methane emission intensity.

Discussion

Line 263: I do not think this is the best way to discuss such a complex relationship.

Line 298: So, the forage type alters milk and fatty acid composition? The diverse pasture is better than the other two pastures? I believe this question deserves further discussion.

Line  385: Authors make excessive reference to Bougouin et al. paper. They must include other papers in the discussion. Authors acknowledge that Bougouin paper is more extensive and includes a larger number of observations. However, I believe the authors are missing relevant information from similar papers.

Line 389: Mention what are the alternative equations?

Line 401 : hard to-read sentence.

Line 404:  I do not recall studies where milk yield correlates with CH4 daily production. Insert some references to support your assumption.

Line 412: Exactly, authors must focus on the effect of types of cow, forage or production system. They already acknowledge that there is not much case comparing their study with that in Bougouin et al. paper.

Line 426: Authors do not present results for methane emissions. So, its hard to conlude on low methane emissions.

Line 434: Present the methane emissions per treatment and not as average.   

The authors also need to discuss why models overpredict methane emissions. I believe this will be a better discussion instead of insisting in comparing your work with Bougouin paper. These are two different types of paper, one is a meta-analysis and the other is an experiment.

Conclusion

Your results do not support your conclusion. You may also want to conclude on the diverse pasture, which is your objective as indicated in the title.

 

 

Comments for author File: Comments.pdf

Author Response

Thanks you for the extensive feedback on our paper.  I hope the next version is more acceptable, taking on board most of the points you made.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear Editor and Authors,

In my opinion, this revised article incorporates most of the points raised in the original draft. However, a few minor points must still be addressed before the document can be published. Please accept my best wishes for all of the authors who contributed to this wonderful work and best wishes for their future endeavours.

Major comments:

L272-279: In the statistics section, you indicate that you are using the results of a predictive model. As a reader, I would expect to see these results in a table or graphic format. In my opinion, as a referee, I would like to see clearly the results of a parameter for which you provide materials and methods. Consequently, these values, as well as means and standard deviations of the recorded values, should be presented.

Table 2a and 2b: In light of the explanations you have provided, it would be helpful to indicate this situation by at least changing the names of the tables.

Figure 2: The inconsistency between "herbs" (in figures) and "diverse sward" (in tables) should be fixed.

Comments for author File: Comments.docx

Author Response

In my opinion, this revised article incorporates most of the points raised in the original draft. However, a few minor points must still be addressed before the document can be published. Please accept my best wishes for all of the authors who contributed to this wonderful work and best wishes for their future endeavours.

Thank you for these kind words – I am sure they will inspire my younger co-authors

Major comments:

L272-279: In the statistics section, you indicate that you are using the results of a predictive model. As a reader, I would expect to see these results in a table or graphic format. In my opinion, as a referee, I would like to see clearly the results of a parameter for which you provide materials and methods. Consequently, these values, as well as means and standard deviations of the recorded values, should be presented.

As you suggest, these results are now presented as Table 2, rather than explained in the text.

Table 2. Measured methane output compared with model predictions, using equations from Bougouin et al. [1] for a) dairy output (g/cow/day) and b) relative to milk yield (g/kg energy corrected milk) (means ± SEM)
a)      daily output		b)      relative to yield
measured	253 ± 9.6	measured	8.7 ± 0.33
model 1	476 ± 4.4	model 15	12.2 ± 0.08
model 6	365 ± 3.3	model 17	17.2 ± 0.34

 

Table 2a and 2b: In light of the explanations you have provided, it would be helpful to indicate this situation by at least changing the names of the tables.

These have become Tables 3a & b and the titles now reads as “Table 3a Outcome of correlation analysis using fatty acid profiles (% total fatty acids) and recorded methane emissions (g/cow /day) (showing those with P-values <0.05)”  and “Table 3b Outcome of correlation analysis using fatty acid profiles (% total) and recorded methane emissions relative to yield (g/kg energy corrected milk) (showing those with P-values <0.05)”

 

Figure 2: The inconsistency between "herbs" (in figures) and "diverse sward" (in tables) should be fixed.

Apologies, both charts have been replaced with ‘diverse’ in instead of ‘herbs’

 

Author Response File: Author Response.docx

Reviewer 2 Report

After reviewing the document, the authors did not make some comments because the other reviewers had not asked for them, so the changes were minimal to enrich the document. 

Author Response

Apologies but, since this reviewer gave scant detail on how our manuscript could be improved, we were at a loss to implement their guidance.  I hope the current version is more acceptable.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors of the manuscript addressed most of my questions and concerns. However, there is one key point that was not sufficiently clarified and justified. The duration of the experimental periods is too short even if this approach was already published. In all similar experiments, the duration of experimental periods is at least 21 days, where 14 days are used for diet adaptation and rumen washout. There is an enormous amount of literature that explains the reason to do this. So, the authors need to provide a better justification supported by the necessary references. Also, it is not sufficiently explained why prediction models overestimate methane emissions.

Author Response

The authors of the manuscript addressed most of my questions and concerns. However, there is one key point that was not sufficiently clarified and justified. The duration of the experimental periods is too short even if this approach was already published. In all similar experiments, the duration of experimental periods is at least 21 days, where 14 days are used for diet adaptation and rumen washout. There is an enormous amount of literature that explains the reason to do this. So, the authors need to provide a better justification supported by the necessary references.

 

There are 2 issues questioned by the comment above relating to the trial duration – initially, the period of adaptation prior to data collection but also the duration of methane collection itself. 

1. We recognize the importance of allowing the rumen microbiota to adapt to dietary changes before monitoring any consequences. However, under the conditions of this particular study, we feel 4 days was adequate prior to methane collection starting, giving 8 days before milk sampling for fatty acids, for the following reasons: a) as stated, all cows had 5 weeks (35 days) alternating on both swards prior to the trial to allow rumen microbiome adaptation, b) diet compositions were NOT dissimilar for both ‘treatments’ – concentrate feeds (2kg/head) were common and although the diverse sward did have some herb present, these only contributed 23% of total dry matter, c) subtle changes in diet have been shown to influence rumen metabolites but not the microbiota (see O’Callaghan et al 2018 Metabolites 27) and finally d) in this paper we compare milk fatty acid profiles but NOT methane produced from the different swards and 8 days is more than adequate for changes in fat composition (see Stergiadis et al 2014, Food Chemistry, 164).
2. With respect to the duration of methane collection, many published studies report methane collection for less than the 7 days implied above. The review of protocols for methane collection by Della Rossa, (Animal Feed Science and Technology 2021) includes 45 studies using SF6 where collection was between 2 and 4 days (in addition to over 200 respiration chamber trials lasting 4 days or less).

We modified the text, both in M&M, hopefully to explain why this procedure was followed.  

“Cows (paired for milk yield, parity, liveweight and calving date) grazed allocated pastures for 4 days, ahead of 4 days methane assessment, with milk for FA determination collected on the last day - before being repeated on the other pasture type.  As stated by Loza et al [2] this relatively short period of adaptation was judged adequate under the circumstances, since all cows (or their rumen microbiota) were exposed to both pasture types for 5 week prior to recording and the resulting 8 day was more than sufficient to stabilise milk fatty acids [Stergiadis, et al. [3].  Also, since concentrate feeding was common on both treatments, the relatively minor adjustments in diet composition from grazed forage during the 4 day change-over, are unlikely to influence rumen microbiota [O’Callaghan, et al. [4]….”

Also, it is not sufficiently explained why prediction models overestimate methane emissions.

The relatively low methane measurements in this study compared with other work is already well discussed in the paper by Loza et al.  So, since this is the driver of the poor prediction from existing models for this data set, we are unsure this is an appropriate forum to discuss this again.  We do suggest the following modifications to the manuscript (around line 700 in the latest version), which hopefully flags up the low methane records and hence the weakness of applying prediction models :

“Methane measurements reported (and discussed) by Loza, tended to be lower than most other published work, especially for ‘intensity’ when compared relative to the high milk output recorded in this study.  As such, there are consistent discrepancies between our 40 records when compared to methane predictions from models based on over 800 records in the meta-analysis by Bougouin et al. [1].  Despite ignoring positive contributions linked to iso-C16:0 (not quantified in our study) from calculations for equations 15 and 17, predicted methane outputs were all higher than measured values, by an average of 29-30% for models 6 and 15 with values of 47 and 49% higher respectively for models 1 and 17.  Consequently, stage 2 was implemented, to identify correlating FA then investigate alternative regression equations which might be more appropriate for this dataset from Jersey cows under organic management, grazing high quality forages”

Author Response File: Author Response.pdf