Comparative Accuracy of In Vitro Rumen Fermentation and Enzymatic Methodologies for Determination of Undigested Neutral Detergent Fiber in Forages and Development of Predictive Equations Using NIRS
, Eun-Chan Jeong 2, Li-Li Wang 2, Rajaraman Bharanidharan 3 and Jong-Geun Kim 1,2,*
Round 1
Reviewer 1 Report
Dear Authors
Reviewer(s)' Comments to Author:
Article: Comparative accuracy of in vitro rumen fermentation and enzymatic methodologies in determination of undigested neutral detergent fiber in forages and the development of predictive equations in selected hays using NIRS
Comments: I have some suggestions as following:
Abstract
Line 36-38: please you give more the interesting advantage
Line 37: please add as a fast screening …….
Line 38: should be delete “the” >>>> important in commercial……
Key words
Line 39: Why type unavailable and not undigested, it should be undigested
Introduction:
Line 73: What you mean about cost? Please explain it.
Line 83: should be change “Past studies……” to “Previous studies……”
Materials and Methods
Line 110: should be change “2” to “two runs….”. and delete 2 run
Line 119: 1-mm screen
Line 125: add sodium sulfite (Na2SO3)
Line 126: adjust to 0.5 g (dry matter)
Line 128: add sulphuric acid (H2SO4)
Line 147: How much volume the jar contains?
Line 154: You already add in the line 123 but have to add USA too then Line 154 should be deleted (Ankom Technologies, Inc., Fairport, NY, USA).
Line 162: preincubation should be change to pre-incubation
Line 166: have to add chemical formular potasium phosphate (H3PO4, 3.56 g/L) and sodium phosphate (Na3PO4, 0.855 w/w)
Line 173 -174: should be use only chemical formular H3PO4 and Na3PO4
Result and Discussion
Line 259: check through manuscript in the word “in vitro” have to use italic “in vitro”
Line 279: “with 72 %H2SO4 ”
Line 280: should be add equation too (how to calculate)
Line 292: add “II” after “incubator”
Line 322: pore size = 25 µm >>> please check through manuscript, should be change (pore size =….. µm) to (……µm pore size)
Line 429 : please check “Ankom DaisyII incubator” have to use the same though manuscript for “Ankom DaisyII incubator” or “Ankom DaisyII incubator”
Line 449: what do you mean “pool size of uNDF” please you explain
Author Response
Dear Editor of Agriculture,
Manuscript ID: agriculture-1955717
Thank you for your letter requesting revisions of our manuscript entitled: “Comparative accuracy of in vitro rumen fermentation and enzymatic methodologies in determination of undigested neutral detergent fiber in forages and the development of predictive equations in selected hays using NIRS”. We have thoroughly revised the manuscript and provided the detailed responses to the comments of the reviewers as described below. We made corrections in the manuscript file in yellow highlight. Therefore, the corrections and improvements can easily be tracked.
We hope that you find the manuscript improved by the revisions and is now acceptable for publication in Agriculture.
Best regards.
Jong Geun Kim (Corresponding author).
Article: Comparative accuracy of in vitro rumen fermentation and enzymatic methodologies in determination of undigested neutral detergent fiber in forages and the development of predictive equations in selected hays using NIRS
Comments: I have some suggestions as following:
AU: Thank you for your constructive review and positive feedback that resulted in improvement of the quality of manuscript.
Abstract
Line 36-38: please you give more the interesting advantage
AU: As suggested the sentence was expanded and revised as follows:
“Although the NIRS equations developed using alfalfa hay dataset were more accurate than that of timothy hay and tall fescue straw, the validation results verified applicability of the equations as a fast screening tool for qualitative pre-diction of uNDF in these forages, which is important in commercial settings”.
Line 37: please add as a fast screening…
AU: Corrected.
Line 38: should be delete “the” >>>> important in commercial…
AU: Corrected.
Key words
Line 39: Why type unavailable and not undigested, it should be undigested
AU: Corrected. It was revised to “undigested neutral detergent fiber”
Introduction:
Line 73: What you mean about cost? Please explain it.
AU: Usually the canulated animals are non-lactating cows or old cows/steers with low productivity that have already been selected for culling from the herd. In most cases, the feed cost over production and profitability is not justified economically to keep them in the herd. From these points, it is usually costly to maintain the canulated animals in a herd.
Line 83: should be change “Past studies……” to “Previous studies……”
AU: Corrected.
Materials and Methods
Line 110: should be change “2” to “two runs….”. and delete 2 run
AU: Corrected. It was revised to “two runs”.
Line 119: 1-mm screen
AU: Corrected.
Line 125: add sodium sulfite (Na2SO3)
AU: Corrected.
Line 126: adjust to 0.5 g (dry matter)
AU: Corrected.
Line 128: add sulphuric acid (H2SO4)
AU: Corrected.
Line 147: How much volume the jar contains?
AU: Each jar capacity in Ankom DaisyII incubator was approximately 3 litters.
Line 154: You already add in the line 123 but have to add USA too then Line 154 should be deleted (Ankom Technologies, Inc., Fairport, NY, USA).
AU: Revised as instructed. (Ankom Technologies, Inc., Fairport, NY, USA) was deleted from L154.
Line 162: preincubation should be change to pre-incubation
AU: Corrected.
Line 166: have to add chemical formular potassium phosphate (H3PO4, 3.56 g/L) and sodium phosphate (Na3PO4, 0.855 w/w)
AU: Corrected. The chemical formulas were added as requested.
Line 173 -174: should be use only chemical formular H3PO4 and Na3PO4
AU: Corrected.
Result and Discussion
Line 259: check through manuscript in the word “in vitro” have to use italic “in vitro”
AU: Corrected throughout the manuscript, and italic “in vitro” was used consistently throughout the manuscript.
Line 279: “with 72% H2SO4”
AU: Corrected.
Line 280: should be add equation too (how to calculate).
AU: Corrected. The sentence was expanded and revised as follows:
“Calculated using the equation of Rohweder et al. [74]: [(digestible dry matter × dry matter intake)/1.29], where digestible dry matter was calculated as 88.9 - (0.779 × ADF%), and dry matter intake as 120/(NDF%)”.
Line 292: add “II” after “incubator”
AU: Corrected throughout the manuscript.
Line 322: pore size = 25 µm >>> please check through manuscript, should be change (pore size =….. µm) to (……µm pore size)
AU: Corrected consistently throughout the manuscript.
Line 429: please check “Ankom DaisyII incubator” have to use the same though manuscript for “Ankom DaisyII incubator” or “Ankom DaisyII incubator”
AU: Corrected. Ankom DaisyII incubator was consistently used throughout the manuscript.
Line 449: what do you mean “pool size of uNDF” please you explain.
AU: The NDF pool sizes are terminologies used in the literature to describe the digestion kinetics of NDF in the rumen. In general, fiber digestion in the rumen is described as a four-phase decay curve: lag, fast pool, slow pool and undigested pool. Once digestion begins, the easily digested part of the fiber disappears quickly. This is the fast pool. Next, the rumen microorganisms work on the more resistant material which digests more slowly. This is called the slow pool. There is a portion of the fiber that cannot be digested in the rumen, which is called the undigested (indigestible) pool.
The fast pool is estimated as the amount of NDF that disappears in 30 hours. The second phase of fiber digestion is the slow pool. The slow pool is estimated as the digestion between 30 and 240 hours. To determine the forage energy potential, it is important to determine the undigested (indigestible) pool of the fiber. Regardless of how long this material stays in the rumen, it will not degrade. The best current estimate of undigested (indigestible) NDF is uNDF240.
Dear Editor of Agriculture,
Manuscript ID: agriculture-1955717
Thank you for your letter requesting revisions of our manuscript entitled: “Comparative accuracy of in vitro rumen fermentation and enzymatic methodologies in determination of undigested neutral detergent fiber in forages and the development of predictive equations in selected hays using NIRS”. We have thoroughly revised the manuscript and provided the detailed responses to the comments of the reviewers as described below. We made corrections in the manuscript file in yellow highlight. Therefore, the corrections and improvements can easily be tracked.
We hope that you find the manuscript improved by the revisions and is now acceptable for publication in Agriculture.
Best regards.
Jong Geun Kim (Corresponding author).
Article: Comparative accuracy of in vitro rumen fermentation and enzymatic methodologies in determination of undigested neutral detergent fiber in forages and the development of predictive equations in selected hays using NIRS
Comments: I have some suggestions as following:
AU: Thank you for your constructive review and positive feedback that resulted in improvement of the quality of manuscript.
Abstract
Line 36-38: please you give more the interesting advantage
AU: As suggested the sentence was expanded and revised as follows:
“Although the NIRS equations developed using alfalfa hay dataset were more accurate than that of timothy hay and tall fescue straw, the validation results verified applicability of the equations as a fast screening tool for qualitative pre-diction of uNDF in these forages, which is important in commercial settings”.
Line 37: please add as a fast screening…
AU: Corrected.
Line 38: should be delete “the” >>>> important in commercial…
AU: Corrected.
Key words
Line 39: Why type unavailable and not undigested, it should be undigested
AU: Corrected. It was revised to “undigested neutral detergent fiber”
Introduction:
Line 73: What you mean about cost? Please explain it.
AU: Usually the canulated animals are non-lactating cows or old cows/steers with low productivity that have already been selected for culling from the herd. In most cases, the feed cost over production and profitability is not justified economically to keep them in the herd. From these points, it is usually costly to maintain the canulated animals in a herd.
Line 83: should be change “Past studies……” to “Previous studies……”
AU: Corrected.
Materials and Methods
Line 110: should be change “2” to “two runs….”. and delete 2 run
AU: Corrected. It was revised to “two runs”.
Line 119: 1-mm screen
AU: Corrected.
Line 125: add sodium sulfite (Na2SO3)
AU: Corrected.
Line 126: adjust to 0.5 g (dry matter)
AU: Corrected.
Line 128: add sulphuric acid (H2SO4)
AU: Corrected.
Line 147: How much volume the jar contains?
AU: Each jar capacity in Ankom DaisyII incubator was approximately 3 litters.
Line 154: You already add in the line 123 but have to add USA too then Line 154 should be deleted (Ankom Technologies, Inc., Fairport, NY, USA).
AU: Revised as instructed. (Ankom Technologies, Inc., Fairport, NY, USA) was deleted from L154.
Line 162: preincubation should be change to pre-incubation
AU: Corrected.
Line 166: have to add chemical formular potassium phosphate (H3PO4, 3.56 g/L) and sodium phosphate (Na3PO4, 0.855 w/w)
AU: Corrected. The chemical formulas were added as requested.
Line 173 -174: should be use only chemical formular H3PO4 and Na3PO4
AU: Corrected.
Result and Discussion
Line 259: check through manuscript in the word “in vitro” have to use italic “in vitro”
AU: Corrected throughout the manuscript, and italic “in vitro” was used consistently throughout the manuscript.
Line 279: “with 72% H2SO4”
AU: Corrected.
Line 280: should be add equation too (how to calculate).
AU: Corrected. The sentence was expanded and revised as follows:
“Calculated using the equation of Rohweder et al. [74]: [(digestible dry matter × dry matter intake)/1.29], where digestible dry matter was calculated as 88.9 - (0.779 × ADF%), and dry matter intake as 120/(NDF%)”.
Line 292: add “II” after “incubator”
AU: Corrected throughout the manuscript.
Line 322: pore size = 25 µm >>> please check through manuscript, should be change (pore size =….. µm) to (……µm pore size)
AU: Corrected consistently throughout the manuscript.
Line 429: please check “Ankom DaisyII incubator” have to use the same though manuscript for “Ankom DaisyII incubator” or “Ankom DaisyII incubator”
AU: Corrected. Ankom DaisyII incubator was consistently used throughout the manuscript.
Line 449: what do you mean “pool size of uNDF” please you explain.
AU: The NDF pool sizes are terminologies used in the literature to describe the digestion kinetics of NDF in the rumen. In general, fiber digestion in the rumen is described as a four-phase decay curve: lag, fast pool, slow pool and undigested pool. Once digestion begins, the easily digested part of the fiber disappears quickly. This is the fast pool. Next, the rumen microorganisms work on the more resistant material which digests more slowly. This is called the slow pool. There is a portion of the fiber that cannot be digested in the rumen, which is called the undigested (indigestible) pool.
The fast pool is estimated as the amount of NDF that disappears in 30 hours. The second phase of fiber digestion is the slow pool. The slow pool is estimated as the digestion between 30 and 240 hours. To determine the forage energy potential, it is important to determine the undigested (indigestible) pool of the fiber. Regardless of how long this material stays in the rumen, it will not degrade. The best current estimate of undigested (indigestible) NDF is uNDF240.
Dear Editor of Agriculture,
Manuscript ID: agriculture-1955717
Thank you for your letter requesting revisions of our manuscript entitled: “Comparative accuracy of in vitro rumen fermentation and enzymatic methodologies in determination of undigested neutral detergent fiber in forages and the development of predictive equations in selected hays using NIRS”. We have thoroughly revised the manuscript and provided the detailed responses to the comments of the reviewers as described below. We made corrections in the manuscript file in yellow highlight. Therefore, the corrections and improvements can easily be tracked.
We hope that you find the manuscript improved by the revisions and is now acceptable for publication in Agriculture.
Best regards.
Jong Geun Kim (Corresponding author).
Article: Comparative accuracy of in vitro rumen fermentation and enzymatic methodologies in determination of undigested neutral detergent fiber in forages and the development of predictive equations in selected hays using NIRS
Comments: I have some suggestions as following:
AU: Thank you for your constructive review and positive feedback that resulted in improvement of the quality of manuscript.
Abstract
Line 36-38: please you give more the interesting advantage
AU: As suggested the sentence was expanded and revised as follows:
“Although the NIRS equations developed using alfalfa hay dataset were more accurate than that of timothy hay and tall fescue straw, the validation results verified applicability of the equations as a fast screening tool for qualitative pre-diction of uNDF in these forages, which is important in commercial settings”.
Line 37: please add as a fast screening…
AU: Corrected.
Line 38: should be delete “the” >>>> important in commercial…
AU: Corrected.
Key words
Line 39: Why type unavailable and not undigested, it should be undigested
AU: Corrected. It was revised to “undigested neutral detergent fiber”
Introduction:
Line 73: What you mean about cost? Please explain it.
AU: Usually the canulated animals are non-lactating cows or old cows/steers with low productivity that have already been selected for culling from the herd. In most cases, the feed cost over production and profitability is not justified economically to keep them in the herd. From these points, it is usually costly to maintain the canulated animals in a herd.
Line 83: should be change “Past studies……” to “Previous studies……”
AU: Corrected.
Materials and Methods
Line 110: should be change “2” to “two runs….”. and delete 2 run
AU: Corrected. It was revised to “two runs”.
Line 119: 1-mm screen
AU: Corrected.
Line 125: add sodium sulfite (Na2SO3)
AU: Corrected.
Line 126: adjust to 0.5 g (dry matter)
AU: Corrected.
Line 128: add sulphuric acid (H2SO4)
AU: Corrected.
Line 147: How much volume the jar contains?
AU: Each jar capacity in Ankom DaisyII incubator was approximately 3 litters.
Line 154: You already add in the line 123 but have to add USA too then Line 154 should be deleted (Ankom Technologies, Inc., Fairport, NY, USA).
AU: Revised as instructed. (Ankom Technologies, Inc., Fairport, NY, USA) was deleted from L154.
Line 162: preincubation should be change to pre-incubation
AU: Corrected.
Line 166: have to add chemical formular potassium phosphate (H3PO4, 3.56 g/L) and sodium phosphate (Na3PO4, 0.855 w/w)
AU: Corrected. The chemical formulas were added as requested.
Line 173 -174: should be use only chemical formular H3PO4 and Na3PO4
AU: Corrected.
Result and Discussion
Line 259: check through manuscript in the word “in vitro” have to use italic “in vitro”
AU: Corrected throughout the manuscript, and italic “in vitro” was used consistently throughout the manuscript.
Line 279: “with 72% H2SO4”
AU: Corrected.
Line 280: should be add equation too (how to calculate).
AU: Corrected. The sentence was expanded and revised as follows:
“Calculated using the equation of Rohweder et al. [74]: [(digestible dry matter × dry matter intake)/1.29], where digestible dry matter was calculated as 88.9 - (0.779 × ADF%), and dry matter intake as 120/(NDF%)”.
Line 292: add “II” after “incubator”
AU: Corrected throughout the manuscript.
Line 322: pore size = 25 µm >>> please check through manuscript, should be change (pore size =….. µm) to (……µm pore size)
AU: Corrected consistently throughout the manuscript.
Line 429: please check “Ankom DaisyII incubator” have to use the same though manuscript for “Ankom DaisyII incubator” or “Ankom DaisyII incubator”
AU: Corrected. Ankom DaisyII incubator was consistently used throughout the manuscript.
Line 449: what do you mean “pool size of uNDF” please you explain.
AU: The NDF pool sizes are terminologies used in the literature to describe the digestion kinetics of NDF in the rumen. In general, fiber digestion in the rumen is described as a four-phase decay curve: lag, fast pool, slow pool and undigested pool. Once digestion begins, the easily digested part of the fiber disappears quickly. This is the fast pool. Next, the rumen microorganisms work on the more resistant material which digests more slowly. This is called the slow pool. There is a portion of the fiber that cannot be digested in the rumen, which is called the undigested (indigestible) pool.
The fast pool is estimated as the amount of NDF that disappears in 30 hours. The second phase of fiber digestion is the slow pool. The slow pool is estimated as the digestion between 30 and 240 hours. To determine the forage energy potential, it is important to determine the undigested (indigestible) pool of the fiber. Regardless of how long this material stays in the rumen, it will not degrade. The best current estimate of undigested (indigestible) NDF is uNDF240.
Reviewer 2 Report
Comments for author File: 
Comments.docx
Author Response
Dear Editor of Agriculture,
Manuscript ID: agriculture-1955717
Thank you for your letter requesting revisions of our manuscript entitled: “Comparative accuracy of in vitro rumen fermentation and enzymatic methodologies in determination of undigested neutral detergent fiber in forages and the development of predictive equations in selected hays using NIRS”. We have thoroughly revised the manuscript and provided the detailed responses to the comments of the reviewers as described below. We made corrections in the manuscript file in yellow highlight. Therefore, the corrections and improvements can easily be tracked.
We hope that you find the manuscript improved by the revisions and is now acceptable for publication in Agriculture.
Best regards.
Jong Geun Kim (Corresponding author).
The objectives of this work were, on the one hand, the comparison between two methods for the determination of the undigested neutral detergent fiber (uNDF) in a set of different forages, by means of in vitro incubation at 240 h following the ANKOM methodology and, on the other hand, an enzymatic method without the use of ruminal fluid. The method that gave the most accurate results (ANKOM in vitro method) was chosen to develop NIRS prediction equations in alfalfa and timothy hay and tall fescue straw. Although the research topic is not excessively novel, especially the one related to the construction of NIRS prediction equations it can add relevant information in the evaluation of methods to determine parameters (uNDF in this case) that are related to the nutritional value of forages.
AU: We would like to thank you for taking the time to provide constructive comments and suggestions on the manuscript. We believe the comments suggested have led to the significant improvement of the manuscript.
Specific Comments
Can you provide more information on the origin and acquisition of the forages used for experiment 1?
AU: The forages in experiment 1 were chosen based on their availability in the forage laboratory and requests to forage importing companies. Unfortunately, the specific information on individual samples such as location, vegetative stage, and harvest date,... was not always accurately available in this experiment (for example in those imported forage samples, information about the agronomic conditions was not easily available), and thus we are unable to report those information.
What was the size of the samples and the 27 subsamples?
AU: The forages in experiment 1 for both chemical composition, in vitro ruminal fermentation method, and also enzymatic method, were hammer-milled and passed through a 1-mm screen size. This information was highlighted in section “2.1. Sample preparation”.
In vitro incubations
From the information given by the authors, I deduce that in each incubation the 6 forages were tested and with 6 replicates/forages in total they had 36 ANKOM F57 bags. Taking into account that in each bottle it is recommended to put 24 bags + 1 blank. The questions are: how these 36 bags were distributed in the bottles??? Was the variability due to the position of each bottle within the incubator considered?
AU: Here we need to clarify that at the time this experiment’s samples (n = 36) were run in Ankom DaisyII incubator, there were additional samples from other experiments in the laboratory. Therefore, complete sets of 24 bags per jar (22 bags containing samples plus 2 blank bags) were put into each jar and placed randomly into ANKOM Daisy Incubators (ANKOM Technology). Also, to account for the variability in within-unit position, the forage types were evenly distributed in 4 rotating jars in each run. This information was added to the main text with yellow highlight.
L141-142 Can you provide more information about the diet of the inoculum donor animals and their distribution? Forage ratio:concentrate? How and when was the food distributed?
AU: More supplementary information were provided, and the text was revised as “Steers were fed twice daily (08:00 and 16:00 h) with rice straw (75% NDF) and a commercial concentrate mix (15.4% crude protein, 29.2% NDF, 13.2% ADF, and 5.24% crude fat).”
L143 I assume that the thermos flasks were kept at 39 ºC until just before introducing the ruminal fluid.
AU: Exactly. The thermos flasks were kept warm (39°C) before the ruminal fluid was added. The following sentence was added to the main text:
“The thermos flasks were kept at 39°C until the ruminal fluid was introduced”.
L154-156 In reference [34] the authors do not use sodium sulfite for the determination of ADL that they do sequentially NDF-ADF-ADL. According to the procedure established by MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002: The use of sodium sulfite has the potential to remove phenolic compounds, but its inclusion is nevertheless important to remove proteins bound to fiber, especially in foods that have undergone some thermal treatment. I do not consider that this is an issue that has had a significant impact on the results obtained, but the effect of sodium sulfite on the estimates of NDF is considered, but not on the protein bound to the fiber, which would be in the opposite direction.
AU: We agree with the reviewer comment that omitting sodium sulfite has an effect on solubilization of bound proteins during the NDF procedure. In this experiment, we followed a reliable reference for determination of uNDF (Raffrenato et al., 2019). In this reference, the authors also omitted sodium sulfite from NDF procedure for the more accurate determination of uNDF fraction in forages.
Raffrenato, E., Nicholson, C.F. and Van Amburgh, M.E., 2019. Development of a mathematical model to predict pool sizes and rates of digestion of 2 pools of digestible neutral detergent fiber and an undigested neutral detergent fiber fraction within various forages. J. Dairy Sci. 102:351-364.
Also, we have to mention that inclusion of Na2SO3 during NDF analysis is a debated matter in the literature. Van Soest (2015) omitted Na2SO3 from his simple NDF procedure, and Van Soest and Robertson (1980) advised how Na2SO3 attacks lignin and causes significant losses to recovery values (Robertson, 1978; Van Soest, 1994).
Regarding the reference [34], in the present manuscript we aimed to refer to the information/explanations provided by the paper from Van Soest et al. (1991) that how sodium sulfite attacks lignin components and may results in lower lignin recovery. Below is the exact extracted paragraph from the original paper (Van Soest et al., 1991):
“The use of sodium sulfite in the NDF procedure remains optional. Its purpose is to lower the protein level and remove keratinaceous residues of animal origin. Sulfite cleaves disulfide bonds and thus dissolves many crosslinked proteins. Its general use for ruminant feeds is discouraged, especially if the residues are to be used as an assay for ND insoluble protein, because the sulfite reaction is nonbiological. The ND and acid detergent insoluble proteins from animal products tend to be indigestible. Sulfite also attacks lignin and therefore should not be used in sequential analyses leading to lignin determination or when the residue is to be used for subsequent in vitro digestion with rumen organisms”.
Multi-step enzymatic method
There are some aspects of this procedure that I think the authors take for granted and that require explanation and, where appropriate, correction.
Since the forage samples were weighed into the same F57 bags, incubation took place in the DaisyII incubator bottles?
AU: In multi-step enzymatic method, the 0.3-g forage samples were weighed into Ankom F57 filter bags, according to the procedure of Gallo et al. (2019). But the enzymatic method was not performed in the DaisyII incubator.
As I seem to understand the steps followed were Immersion for 90 min of the ANKOM bags in a 0.2 M NaOH solution with a ratio of 15 mL of solution per bag. After washing the bags, they were incubated for 24 h at 39 ºC in a buffer solution to which the next enzymes were added: cellulases, hemicellulases and Viscozyme in a ratio of 15 mL/L??? won't it be 15mL/bag???
AU: Thank you for catching up this inadvertent error. It was “15 mL/bag” and corrected in the revised text.
Idem for the next step
I assume that the determination of the NDF residue from this incubation was done the same as in the in vitro determination.
Spectra acquisition and calibration development
Was the chemical composition and uNDF determined for all samples? Can you explain how the uNDF was determined for the 254 samples??? Specifically number of runs, number of replicates of each sample per run, how the position of the bottle was taken into account in each run, etc.
AU: In experiment 1, in vitro ruminal fermentation using Ankom Daisy incubator was selected as the most accurate reference method for the uNDF to construct predictive equations for 21 uNDF estimation using near-infrared reflectance spectroscopy. This information was highlighted in abstract section as well as in section 2.5. Spectra acquisition and calibration development.
The chemical composition (DM, NDF, ADF, ADL, crude protein, and calculated relative feed value) and uNDF were determined for all samples in experiment 2 (n = 264) including alfalfa and timothy hays, and tall fescue straw.
To clarify the methodology, the following text was added:
AU: The in vitro experiment was performed using Ankom DaisyII incubator in two runs with two replications of each sample in each run.
Also, to account for the variability in within-unit position, the forage types were distributed in four rotating jars in each run.
The cross-validation with which subsample was done?
AU: Cross-validation statistics were generated in the software UCalTM ver. 4.0 (Unity Scientific of KPM Analytics, USA) using the input data of forage samples including alfalfa and timothy hays, and tall fescue straw.
Data analysis
L211- The comparison between the two methods was carried out using the Tukey?? Since only two methods were tested, it would not be necessary to carry out a test to compare means. What model did you use for the analysis of variance?
AU: Thank you for the comment. We consulted with a statistician in the field about the accuracy of the procedure for the statistical analysis, in particular the data analysis in Table 2. As we presented the results and discussion about the accuracy of the method (enzymatic vs. in vitro ruminal fermentation), we needed to compare the means of each forage type by method of determination (enzymatic vs. in vitro method). Actually, the Tukey test was used to compare the difference in uNDF concentration of each forage type by the method of measurement. And the overall difference was evaluated using Levene’s test. Alternatively, we could have also adopted another statistical analysis procedure using a model including the method, the forage type, and the interaction between method and forage, as fixed effects, and incubation run as a random variable. However, we believe the procedure currently used in the manuscript provides a clear description on which method better determines uNDF concentration in each forage type.
L258-259 re-write this sentence. Something like “Except for alfalfa hay, the uNDF values obtained with the enzymatic method were higher than those of the in vitro method (P < 0.01)”
AU: As recommended, the sentence was revised to:
“Except for alfalfa hay, the uNDF values obtained with the enzymatic method were higher than those of the in vitro method (P < 0.01)”.
L283 summary??
AU: It was corrected to “Based on the statistics”.
Relationship between ADL and uNDF
I do not understand very well what this section contributes to the objectives set by the authors. Neither in the introduction nor in M&M is there any reference to this section. Besides, the discussion is basically focused on the possible escape of plant material from the samples through the pores of the bag. The authors have not considered other factors such as the underestimation of the lignin content when estimating it by the ADL methodology, especially in grasses. See the works of Fukushima RL in this regard.
AU: We agree with the reviewer comment that we could have provided some information before in introduction or M&M. The second objective of experiment 2 in introduction section was revised as follows:
“In experiment 2, the objective was to employ the most accurate reference method selected in experiment 1 for uNDF quantification of a large pool of alfalfa and timothy hays, as well as tall fescue straw, and assess the accuracy of predicting uNDF component using acid deter-gent lignin (ADL) parameter and predictive equations using NIRS”.
As suggested by the reviewer, we added the following paragraph to the revised text, using the results extracted from Fukushima’s works. The following paragraph was added as follows:
“The laboratory method of lignin determination is another important factor that needs to be taken into consideration. Fukushima et al. [65] identified that the ADL method (using concentrated sulfuric acid) provided an underestimation of lignin concentration in grasses when compared with the spectrophotometric acetyl bromide lignin method. Hemicellulose solubilization during the ADF treatment was suggested to leave lignin components in a configuration that facilitates the detachment of some lignin groups, thereby resulting in lignin solubilization and loss during the ADL procedure [66,67]. Further research into the comparative evaluation of lignin determination methods (ADL vs. acetyl bromide lignin method) may result in a better establishment of the relationship between ADL and uNDF”.
In the uNDF vs. ADL graphs, indicate which forage corresponds to each graph. It has only been indicated for tall fescue straw.
AU: Sorry for this error. Each forage corresponding to each graph was already provided in Word file in the original submission, but it seems it was hidden when a pdf file was created during the submission process. They are now visualized beside each graph.
Author Response File: Author Response.docx
