Techniques Used to Determine Botanical Composition, Intake, and Digestibility of Forages by Ruminants

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Review on “Techniques used to determine botanical composition, intake, and digestibility of forages by ruminants”

 

General comments

I have only minor comments on this Review.

Authors need to make it clear when they are referring to techniques to estimate total diet DM intake (or digestibility), or forage DM intake, or the actual nutrient intake when describing each technique during the abstract, introduction and other sections.

 

Specific comments

Abstract

L17: add comma after limitation

L19: total diet intake? This Review is not about forage intake?

 

1.       Introduction

L34. Include a dot after the citation.

 

2.       Methods of estimating the botanical composition of selected diets

Add somewhere in this section what authors defined as botanical composition.

L156-158: The stomach content method has a lot of limitations and should not be used to validate other techniques. What is the gold standard method to evaluate the botanical composition of forage ingested by animals?

 

3.       Analytical procedures in estimating diet intake in grazing animals

L176: remove extra space after citation 33

L189: internal markers are extensively used to estimate fecal output in confined cattle.

L218: please, check the extra spaces throughout the manuscript.

L222: remove “a” after “only”

 

L268: add unit for KSI

Author Response

Reviewer 1

Major comments
Authors need to make it clear when they are referring to techniques to estimate total diet DM intake (or digestibility), or forage DM intake, or the actual nutrient intake when describing each technique during the abstract, introduction and other sections.	This has been noted and corrected across the manuscript.
Minor comments
Abstract
L17: add comma after limitation	Comment accepted and correction done
L19: total diet intake? This Review is not about forage intake?	Comment accepted and correction done
Introduction
L34. Include a dot after the citation	Comment accepted and correction done
Methods of estimating the botanical composition of selected diets	Comment accepted and correction done
Add somewhere in this section what authors defined as botanical composition.	This has been updated. See L61
L156-158: The stomach content method has a lot of limitations and should not be used to validate other techniques. What is the gold standard method to evaluate the botanical composition of forage ingested by animals?	The plant cuticular marker technique has been reported to have a high success rate in prediction of the diet selection in animal nutrition trials. However, the cost associated with the technique limits its application, necessitating the quantification of the magnitude of error from stomach analysis in wild and domesticated ruminants killed by predators or slaughtered for human consumption, respectively.  Such, would be to essentially define the stomach content analysis predictive ability to predict diet selection in ruminants, given the economic tradeoffs associated with the technique.
Analytical procedures in estimating diet intake in grazing animals	Correction done
L176: remove extra space after citation 33	Done
L189: internal markers are extensively used to estimate fecal output in confined cattle.	The authors noted that internal markers can be used in both confined and grazing ruminants-cattle, sheep etc. No action taken.
L218: please, check the extra spaces throughout the manuscript.	Done
L222: remove “a” after “only”	Done
L268: add unit for KSI	% inserted.

 

Reviewer 2 Report (New Reviewer)

This study revises the main methodologies that can be used to assess diet composition, feed intake and digestibility of forages by ruminant species. This subject falls within the scope of Agronomy journal and reveals innovation and it would be of interest to the readership. For these reasons, this manuscript should be accepted after revision of some points. Manuscript is well written and very easy to follow and the reference list include the main publications on this subject. However, I’ve some questions, comments and also some suggestions that can improve the quality of the manuscript:

 

MAIN COMMENTS

1) One technique that can be used to evaluate diet selection in all herbivore species and it is now applied in very different situations is the DNA barcoding. The results are promising due to its application in very complex situations where the number of plant species to be identified is very large. The inclusion of a point on this subject will increase the quality of the this review article;

2) In point 2.3. Microhistological procedures, it is referred that “An important drawback of the stomach content method is the variation in the degradability of the plant species consumed” and the same applies to faecal samples. As a results estimates can be biased towards to diet components of lower digestibility over those with higher digestibility where the number of fragments are lower. Moreover, in these procedures a high number of fragments remain to identify. These should be included on the text. Point 2.4. Faecal analysis technique can be merged with point 2.3.

3) Page 11 lines 490-492: in the least square optimization procedures, two types of data can be used in the calculations and will give different results. Absolute biomarker concentrations will give us contribution of each diet component in the animal diet that is needed to produce 1 kg of faecal dry-matter and, consequently, a diet digestibility estimate can be obtained. If proportions of biomarker concentrations relative to the total concentrations are used that will produce proportions of each diet component considered in the calculations, in this case these results cannot be used to estimate diet digestibility. This should be clarified in the manuscript.

4) Pages 11-12 lines 509-512: possibly the type of dosing of the external marker (i.e. paper pellets, gelatine capsules, controlled release devices, etc) has an effect on the period size to obtain a uniform excretion. A comment on this issue can be incorporated in the text.

5) When using the biomarkers in complex grazing natural pastures in terms of number of possible diet components that exceed the number of markers there are a few approaches that can be used, for example grouping plant species with similar biomarker profiles and the eventual problem of selectivity within the group of plant in the group, combination of techniques to reduce the number of possible diet components, etc that should be included and discussed by the authors.

 

MINOR COMMENTS

1) Throughout the text, alkanes should be replaced by n-alkanes, and “n-alkanes” by “n-alkanes”;

2) “Long-chain” instead of “long chain”;

3) Page 5 line 231 the authors include the reference 8 to the application of long-chain fatty acids and this is not correct. Change the used reference to other related to the subject;

4) Table 2: replace “Culluna” by “Calluna”; Trifolium should be in italic; “Lolium multiflorum” instead of “Loliummultiflorum”; “Lolium perenne and Ulex gallii” instead of “Loliumpereneand Ulex gallii

5) Page 8 lines 318-319: the proportions of a possible diet component considered in the calculations can be equal to zero. Modify to “… and their proportions (p) should be constrained to be 0 < p ≤1 and their sum adds up to 1.”

6) Page 9 line 370: “Plant n-alkane (generally C₃₁ and/or C₃₃) measures digestibility ….., while synthetic orally dosed even-numbered (C₃₂ or C₃₆) …”

7) Equation 5: in general, and that is the greatest advantage of using n-alkanes markers for feed intake estimation, the faecal recoveries of n-alkanes i and j are similar, and for that reason there is no need to correct n-alkane faecal concentrations;

8) More recent works have deal with feed intake using the labelled supplement method, replace the work of Dove and Oliván (1998) for a more recent work.

Author Response

Reviewer two

Minor comments	Response
Throughout the text, alkanes should be replaced by n-alkanes, and “n-alkanes” by “n-alkanes	Done
“Long-chain” instead of “long chain	Done
Page 5 line 231 the authors include the reference 8 to the application of long-chain fatty acids and this is not correct. Change the used reference to other related to the subject	The error is acknowledge. Reference 79 has been correctly inserted.
Table 2: replace “Culluna” by “Calluna”; Trifolium should be in italic; “Lolium multiflorum” instead of “Loliummultiflorum”; “Lolium perenne and Ulex gallii” instead of “Loliumpereneand Ulex gallii	Corrections effected.
Page 8 lines 318-319: the proportions of a possible diet component considered in the calculations can be equal to zero. Modify to “… and their proportions (p) should be constrained to be 0 < p ≤1 and their sum adds up to 1.”	Corrections effected.
) Page 9 line 370: “Plant n-alkane (generally C31 and/or C33) measures digestibility ….., while synthetic orally dosed even-numbered (C32 or C36) …”	Correction effected
Equation 5: in general, and that is the greatest advantage of using n-alkanes markers for feed intake estimation, the faecal recoveries of n-alkanes i and j are similar, and for that reason there is no need to correct n-alkane faecal concentrations;	There is no reason to include recoveries if the n-alkanes used are of adject carbon number but there are or possible instances where researchers might have octacosain (C28) as an external marker and the forage or diet contains only C31/33 as an internal marker there would be high discrepancy in terms of the recovery rates. The equation in its current form caters for such.  The reviewer may wish to provide further clarification.
More recent works have deal with feed intake using the labelled supplement method, replace the work of Dove and Oliván (1998) for a more recent work.	Cottle 2013 has bee cited to replace Dove and Oliván (1998).
Major Comments
“One technique that can be used to evaluate diet selection in all herbivore species and it is now applied in very different situations is the DNA barcoding……”	Section 2.3.2 has been inserted from L 176-208 to highlight the application of \DNA bar coding in diet composition prediction from faecal samples. This is a valuable addition and we thank the reviewer for this.
In point 2.3. Microhistological procedures, An important drawback of the stomach content method is the variation in the degradability of the plant species consumed” and the same applies to faecal samples. As a results estimates can be biased towards to diet components of lower digestibility over those with higher digestibility where the number of fragments are lower. Moreover, in these procedures a high number of fragments remain to identify. These should be included on the text. Point 2.4. Faecal analysis technique can be merged with point 2.3.	This has been updated. See line 146-150.
Page 11 lines 490-492: in the least square optimization procedures, two types of data can be used in the calculations and will give different results. Absolute biomarker concentrations will give us contribution of each diet component in the animal diet that is needed to produce 1 kg of faecal dry-matter and, consequently, a diet digestibility estimate can be obtained. If proportions of biomarker concentrations relative to the total concentrations are used that will produce proportions of each diet component considered in the calculations, in this case these results cannot be used to estimate diet digestibility. This should be clarified in the manuscript.	Amendments were made and the following statement was added; “The absolute concentrations of biomarkers are important in the quantitative estimation of the contribution of each forage component in the animal diet that reflects in the faecal dry-matter and consequently in estimating diet dry matter digestibility using equation (8). Where only proportional concentrations of biomarkers relative to total biomarker concentration are used, the proportion of each diet component is estimated but due varying recovery rate, digestibility estimates would not reflect true digestibility of the diet.
) Pages 11-12 lines 509-512: possibly the type of dosing of the external marker (i.e. paper pellets, gelatine capsules, controlled release devices, etc) has an effect on the period size to obtain a uniform excretion. A comment on this issue can be incorporated in the text.	Dosing frequency, dosing method and the use of encapsulated/slow release techniques have been noted to impact biomarker distribution in the gut. Line 564-566
5) When using the biomarkers in complex grazing natural pastures in terms of number of possible diet components that exceed the number of markers there are a few approaches that can be used, for example grouping plant species with similar biomarker profiles and the eventual problem of selectivity within the group of plant in the group, combination of techniques to reduce the number of possible diet components, etc that should be included and discussed by the authors.	Grouping species does not give a true reflection of the consumed plant species’ per ingredient composition. The principal approach is to identify each plant species selected and consumed by the animal to predict their influence on the animal productivity based on the per-ingredient composition. However, where the possible number of biomarkers is limiting the grouping of plant species in a selected diet is the prudent approach to at least get an insight about the selection choices of group of plant species as the first step. Thereafter, the selected groups might be analysed for their associative/ combined chemical composition to determine the potential nutrient availability to the host animal to match with its nutrient requirements.

 

Round 2

Reviewer 2 Report (New Reviewer)

The authors have introduced most of the proposed changes and suggestions and with this the quality of the work has improved significantly. I have only two comment: 1) The authors have introduced this statement “The absolute concentrations of biomarkers are important in the quantitative estimation of the contribution of each forage component in the animal diet that is reflected in the faecal dry-matter and consequently in estimating diet dry matter digestibility using equation (8). Where only proportional concentrations of biomarkers relative to total biomarker concentration are used, the proportion of each diet component is estimated but due varying recovery rate, digestibility estimates would not reflect true digestibility of the diet.” Is not totally correct as the recovery rates are applied before diet composition estimations either using absolute or relative of the markers. SUGGESTION: statement should be modified to “The absolute concentrations of biomarkers are important in the quantitative estimation of the contribution of each forage component in the animal diet that is reflected in the faecal dry-matter and consequently in estimating diet dry matter digestibility using equation (8). Where only proportional concentrations of biomarkers relative to total biomarker concentration are used, the proportion of each diet component is estimated not allowing the estimation of diet digestibility.”; 2) Table 2: replace “Culluna” by “Calluna”;

Author Response

The authors have noted the additional comments and corrections of the reviewers and these have been corrected accordingly. We are grateful for the meticulous review by the editorial team and the anonymous reviewers. 

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This manuscript is well written and easy to read/follow. 

I just have 2 minor comments - 

Title

I think this review focuses on grazing cattle (not confined) thus, authors should consider adding somewhere in the title “grazing cattle”.

Authors need to be consistent with anything related to intake - always use predicted or estimated intake when it was not directly measured.

 

Reviewer 2 Report

The subject is interesting at different levels but the text is not easy to read and sometime difficult to understand.  The organization of the text is not clear and must be changed.  I think that some parts must be explained and developed (see word file).

Comments for author File: Comments.pdf