Reliability of Acid-Insoluble Ashes and Undigestible Neutral Detergent Fibre as Internal Markers for Estimation of Digestibility in Beef Cattle Fed High-Concentrate Diets
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
- Where is the ingredients and nutrition composition of experimental diet ?, where is animals information (weight, how to treated, etc.) and animal care information? all of this is basic for animal trials. Even this methods was already published in researchers article, author still need to statement (briefly) there own methods.
- Is orts means residual feed of animal？use the academic vocabulary. Why consider orts?
- Figure1, name of Y axis should be “uNDF” not "iNDF", is this figure showed the same results with Table 1?
Author Response
Dear reviewer,
Thank you very much for your encouraging and supporting comments.
Reviewer: Where is the ingredients and nutrition composition of experimental diet ?, where is animals information (weight, how to treated, etc.) and animal care information? all of this is basic for animal trials. Even this methods was already published in researchers article, author still need to statement (briefly) there own methods.
Response: Brief information about animals, animal care, and diets used in the different experiments, has now been included in lines 79-103 of the revised version.
Reviewer: Is orts means residual feed of animal？use the academic vocabulary. Why consider orts?
Response: Yes, the term orts refers to feed residues. It has been changed throughout the text. The use of either internal and external markers rely on the concentration of these in the intake and in the faeces. As the animals may select different proportions of barley and concentrate, the concentration of the markers in the feed residues may be different from that in the offered feedstuffs, and this may alter the concentration of markers in the intake. This was the reason for considering feed residues in a first instance, and comparing the results obtained considering feed residues or not. A couple of sentences pointing out this have been included in L124-128 of the revised version of the manuscript.
Reviewer: Figure1, name of Y axis should be “uNDF” not "iNDF", is this figure showed the same results with Table 1?
Response: The name of axis Y in Figure 1 has been corrected. Table 1 gives the results of the analysis of variance (average values) whereas Figure 1 gives de relationships between digestibility values obtained using different methods at an individual level. Data are related but are not the same.
Should you and the other reviewer be happy with the corrections made to the manuscript, it will be sent to MDPI Author Services which offers language editing.
Kind regards,
Antonio de Vega
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe paper present relevant data. I invite authors to respond to attached comments:
Abstract
Correct to “Digestibility, together with intake, are the main factors affecting …”
In this sentence “the relationships between digestibility val- 21 ues obtained in metabolism cages or using Cr2O3 as an external marker, and AIA or uNDF, 22 were not significant” what do you mean by relationships ? correlation or differences ?
Introduction
What is the difference between “administered in the diet and orally?”
I invite authors to cite other methods for assessing the digestibility of feeds, such as enzymatic methods, gas syringes, and DAISY II incubators. They refer to the attached papers
Boukrouh, S., Noutfia, A., Moula, N., Avril, C., Louvieaux, J., Hornick, J. L., ... & Cabaraux, J. F. (2023). Ecological, morpho-agronomical, and nutritional characteristics of Sulla flexuosa (L.) Medik. ecotypes. Scientific Reports, 13(1), 13300.
Abdillah, A. E., Sarah, D., Ardian, A. A., Anas, M. A., Aprianto, M. A., Hanim, C., ... & Yusiati, L. M. (2024). Effect of nutmeg essential oil (Myristica fragrans Houtt.) on methane production, rumen fermentation, and nutrient digestibility in vitro. Scientific Reports, 14(1), 3554.
Boukrouh, S., Noutfia, A., Moula, N., Avril, C., Louvieaux, J., Hornick, J. L., ... & Chentouf, M. (2023). Ecological, morpho-agronomical, and bromatological assessment of sorghum ecotypes in Northern Morocco. Scientific Reports, 13(1), 15548.
I invite the authors to explicitly state the research gap earlier and more clearly (lack of data on AIA/uNDF use in beef cattle with barley straw diets).
Methodology
Add the ethical statement to the start of the “Methodology” section.
In “2.1. Animals, Diets, and Experimental Procedures” section, can you further detail the methods used for assessing digestibility ?
Add a title for statistical analyses.
Discussion
The paragraphs are densely written, and clarity would benefit from breaking them into smaller sub-sections (e.g., one for AIA results and interpretation and one for uNDF).
The discussion attributes the extreme values of AIA (e.g., 340% recovery) mainly to soil contamination due to outdoor feces collection. While plausible, additional discussion on how this might have been mitigated (e.g., repeated sampling, replicates, or removal of outliers) would strengthen the reliability of the conclusion.
The authors acknowledge switching between Method A and Method B for AIA quantification but found a <10% difference. Still, given the high variation in recoveries (15–340%), it would be worth suggesting more robust marker calibration methods or pre-collection cleaning techniques, especially for outdoor studies.
The text correctly identifies uNDF challenges, such as poor accuracy with finely ground feeds. However, the conclusion that particle size was likely not the main problem should be supported by the data or size distribution measurements of the concentrates used.
The suggestion to improve uNDF determination methods for concentrates is constructive, but the authors should mention recent advances or alternatives, such as filtering protocols or NIRS calibration.
The authors accurately stated that AIA provided mean digestibility values similar to total feces collection, but lacked individual reliability. This distinction is crucial and should be emphasized earlier in the section.
The authors strongly defended the use of Cr₂O₃, especially under proper handling. This is appropriate, but they could have added more nuances regarding its limitations in field use, such as sample timing sensitivity or legal restrictions in some regions due to environmental or health concerns.
The inconsistent performance of AIA and uNDF across Experiments 1–3 weakened their practical application. The authors noted this, but could better summarize contextual factors (e.g., weather, feed types, housing systems) that might help explain the inconsistencies and guide future research.
Conclusion
The conclusion that AIA and uNDF are not recommended for high-concentrate diets when individual accuracy is needed is well supported. However, adding a recommendation hierarchy (e.g., "Cr₂O₃ preferred under controlled sampling; AIA only for mean estimates in clean indoor settings") would make the guidance more actionable.
Author Response
Dear reviewer,
Thank you very much for your encouraging and supporting comments.
Reviewer: Abstract. Correct to “Digestibility, together with intake, are the main factors affecting …”
Response: Corrected (line 9 of the revised version).
Reviewer: Abstract. In this sentence “the relationships between digestibility values obtained in metabolism cages or using Cr2O3 as an external marker, and AIA or uNDF, were not significant” what do you mean by relationships? correlation or differences?
Response: We actually refer to the regressions shown in Tables 2 and 4. We have changed ‘relationships’ with ‘regressions’ in L21 of the revised version of the paper.
Reviewer: Introduction. What is the difference between “administered in the diet and orally?”
Response: Administered in the diet means mixed with this latter, whereas orally refers to dosing the animals directly through the mouth (with the aid of a syringe or an oesophageal tube, for instance).
Reviewer: Introduction. I invite authors to cite other methods for assessing the digestibility of feeds, such as enzymatic methods, gas syringes, and DAISY II incubators. They refer to the attached papers
Boukrouh, S., Noutfia, A., Moula, N., Avril, C., Louvieaux, J., Hornick, J. L., ... & Cabaraux, J. F. (2023). Ecological, morpho-agronomical, and nutritional characteristics of Sulla flexuosa (L.) Medik. ecotypes. Scientific Reports, 13(1), 13300.
Abdillah, A. E., Sarah, D., Ardian, A. A., Anas, M. A., Aprianto, M. A., Hanim, C., ... & Yusiati, L. M. (2024). Effect of nutmeg essential oil (Myristica fragrans Houtt.) on methane production, rumen fermentation, and nutrient digestibility in vitro. Scientific Reports, 14(1), 3554.
Boukrouh, S., Noutfia, A., Moula, N., Avril, C., Louvieaux, J., Hornick, J. L., ... & Chentouf, M. (2023). Ecological, morpho-agronomical, and bromatological assessment of sorghum ecotypes in Northern Morocco. Scientific Reports, 13(1), 15548.
Response: With all due respect, the papers suggested by the reviewer refer to in vitro methods for assessing digestibility, and our focus was in in vivo methods. The term in vivo has been included in L10 and L31 of the revised version of the paper.
Reviewer: Introduction. I invite the authors to explicitly state the research gap earlier and more clearly (lack of data on AIA/uNDF use in beef cattle with barley straw diets).
Response: The lack of data has been included in L54-55, and in L61-64 of the revised version of the paper.
Reviewer: Methodology. Add the ethical statement to the start of the “Methodology” section.
Response: Information about ethical aspects has been included in L86-94 of the revised version of the paper.
Reviewer: Methodology. In “2.1. Animals, Diets, and Experimental Procedures” section, can you further detail the methods used for assessing digestibility?
Response: The required information has been included in L75-76, L77-78, and L79-80 of the revised version of the paper.
Reviewer: Methodology. Add a title for statistical analyses.
Response: It is included in section 2.3. Mathematical and Statistical Methods (L118 of the revised version of the paper).
Reviewer: Discussion. The paragraphs are densely written, and clarity would benefit from breaking them into smaller sub-sections (e.g., one for AIA results and interpretation and one for uNDF).
Response: As requested, more subsections have been added (L251, L290, L312, L348 and L377 of the revised version of the paper).
Reviewer: Discussion. The discussion attributes the extreme values of AIA (e.g., 340% recovery) mainly to soil contamination due to outdoor feces collection. While plausible, additional discussion on how this might have been mitigated (e.g., repeated sampling, replicates, or removal of outliers) would strengthen the reliability of the conclusion.
Response: A statement dealing with how the faeces samples were collected has been included in L283-287 of the revised version of the paper).
Reviewer: Discussion. The authors acknowledge switching between Method A and Method B for AIA quantification but found a <10% difference. Still, given the high variation in recoveries (15–340%), it would be worth suggesting more robust marker calibration methods or pre-collection cleaning techniques, especially for outdoor studies.
Response: We are deeply sorry but do not really understand what the reviewer suggests. As stated in L271-273, the differences in AIA concentrations obtained using either method A or method B were less than 5%, and this was translated in even lower differences between digestibility values (less than 5%) obtained using both methods. What does the reviewer mean with ‘Marker calibration methods’ or ‘pre-collection cleaning techniques’? As indicated, the metabolic cages were outdoors and cleaning of the ground was not possible without removing the faeces.
Reviewer: Discussion. The text correctly identifies uNDF challenges, such as poor accuracy with finely ground feeds. However, the conclusion that particle size was likely not the main problem should be supported by the data or size distribution measurements of the concentrates used.
Response: The concentrates in Experiment 1 were offered in the form of meal, with ingredients ground to pass a 6 mm sieve. For this reason, the issue of the lack of uniformity of particle size was probably less important in the present work as the diets included ca. 90 % concentrate, with less variability across particle sizes than forages. This information has been included in L298-302 of the revised version of the paper.
Reviewer: Discussion. The suggestion to improve uNDF determination methods for concentrates is constructive, but the authors should mention recent advances or alternatives, such as filtering protocols or NIRS calibration.
Response: To our knowledge, NIRS has been used for digestibility estimation (e.g. Peters et al., 2025, citation number 4 in the present paper) but not for assessing concentration of uNDF. A recent paper by Jonker and Della Rosa (2024; Animal Production Science 64, AN2428. Doi:10,107/AN2428) has dealt with the comparison of three filter-bag types for accuracy and precision of in sacco undigestible neutral detergent fibre determination of various dicotyledon forages. However, we decided not discussing this aspect as our focus was on in vivo, not in sacco, use of uNDf, and in animals fed high-concentrate diets, not forages.
Reviewer: Discussion. The authors accurately stated that AIA provided mean digestibility values similar to total feces collection, but lacked individual reliability. This distinction is crucial and should be emphasized earlier in the section.
Response: With due respect, this information is given in the second paragraph of this section, and we do not see how to incorporate it earlier.
Reviewer: Discussion. The authors strongly defended the use of Cr₂O₃, especially under proper handling. This is appropriate, but they could have added more nuances regarding its limitations in field use, such as sample timing sensitivity or legal restrictions in some regions due to environmental or health concerns.
Response: The between- and within-days variability of faecal Cr concentration, of paramount importance in field use, has been previously dealt with in our group (Al Alami et al., 2014). This citation has been included in the present paper (number 44). Reference to concerns over potential carcinogenic properties of Cr2O3, and the health hazard when Cr2O3 is inhaled, is given in citations 36, 37 and 43 of the present paper (L358-262).
Reviewer: Discussion. The inconsistent performance of AIA and uNDF across Experiments 1–3 weakened their practical application. The authors noted this, but could better summarize contextual factors (e.g., weather, feed types, housing systems) that might help explain the inconsistencies and guide future research.
Response: Thank you for your suggestion. A paragraph regarding the aspects you mention has been included in L392-394 of the revised version of the paper.
Reviewer: Conclusion. The conclusion that AIA and uNDF are not recommended for high-concentrate diets when individual accuracy is needed is well supported. However, adding a recommendation hierarchy (e.g., "Cr₂O₃ preferred under controlled sampling; AIA only for mean estimates in clean indoor settings") would make the guidance more actionable.
Response: Thanks again. Your suggestion has been incorporated in L400-402 of the revised version of the paper.
Kind regards,
Antonio de Vega
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsAuthors responded to all my questions
