Next Article in Journal
Toward Sustainable Broiler Production: Evaluating Microbial Protein as Supplementation for Conventional Feed Proteins
Previous Article in Journal
A Knowledge-Driven Smart System Based on Reinforcement Learning for Pork Supply-Demand Regulation
Previous Article in Special Issue
Effects of Low-Protein Diet Supplemented with Fermented Feed on Meat Quality, Fatty Acid Composition, and Gut Microbiota in Growing–Fattening Pigs
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agriculture
Volume 15
Issue 14
10.3390/agriculture15141485
agriculture-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Reliability of Acid-Insoluble Ashes and Undigestible Neutral Detergent Fibre as Internal Markers for Estimation of Digestibility in Beef Cattle Fed High-Concentrate Diets

by
Amira Arbaoui
and
Antonio de Vega
*
Departamento de Producción Animal y Ciencia de los Alimentos, Instituto Agroalimentario de Aragón (IA2), Universidad de Zaragoza-CITA, Miguel Servet 177, 50013 Zaragoza, Spain
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(14), 1485; https://doi.org/10.3390/agriculture15141485
Submission received: 12 June 2025 / Revised: 2 July 2025 / Accepted: 7 July 2025 / Published: 10 July 2025
(This article belongs to the Special Issue Assessment of Nutritional Value of Animal Feed Resources)
Browse Figure
Review Reports Versions Notes

Abstract

Digestibility, together with intake, is the main factor affecting animal productivity. It can be assessed in vivo by measuring total feed intake and faecal output (time-consuming and labour-intensive) or with the aid of substances known as markers. Internal markers such as acid insoluble ash (AIA) or undigestible neutral detergent fibre (uNDF) have been alleged to be preferable for digestibility estimations. The use of AIA and uNDF for digestibility estimation in beef cattle fed high-concentrate and barley straw diets has been rarely documented; hence, the objectives of the present paper were to compare digestibility values obtained by total faecal collection vs. AIA or uNDF (Experiment 1), to compare digestibility values obtained using Cr2O3 as an external marker vs. AIA or uNDF (Experiment 2), and to compare digestibility values obtained using AIA vs. uNDF in beef cattle fed high-concentrate and barley straw diets (Experiment 3). Faecal recoveries of AIA and uNDF (Experiment 1) were very variable and likely influenced by contamination of faeces and/or feedstuffs with soil and/or dust. Then, the regressions between digestibility values obtained in metabolism cages or using Cr2O3 as an external marker and AIA or uNDF were not significant. The use of these two latter markers for estimation of digestibility in beef cattle fed high-concentrate and barley straw diets is not recommended.

1. Introduction

Animal productivity is mainly driven by nutrient availability, which in turn depends on both intake and diet digestibility [1,2,3,4,5]. Conventionally, in vivo digestibility has been assessed by measuring total feed intake and faecal output, but this method requires specialised animal handling facilities, is time-consuming, labour-intensive, and costly, and might not be possible in determined conditions such as grazing. The use of markers to determine digestibility overcomes the need to make exact measurements of feed intake and total faecal output [2]. Several internal (those that are naturally present in the feedstuffs, such as lignin [6], chromogen [7], potentially undigestible cellulose and acid-insoluble ash [8], undigestible acid detergent fibre [8], or n-alkanes [9]) and external markers (e.g., chromium [10] or ytterbium [11]) have been evaluated, each with its odds. External markers must be administered in the diet, orally or intraruminally [4], and the diurnal pattern of excretion and unreliable adherence to feed particles may negatively affect their accuracy [3]. For these reasons, internal markers such as acid-insoluble ashes (AIA) or undigestible neutral detergent fibre (uNDF) have been alleged to be preferable for digestibility estimations.
Acid-insoluble ash has been extensively and satisfactorily used in non-ruminant species [2], but its use has been much less reported in ruminants [2,3,5]. Among these, AIA has been employed as a digestibility marker in beef cattle fed mainly forages [3,5,12,13] with reports regarding its use in animals fed high-concentrate diets being practically unavailable [3,5]. Moreover, the two found papers reporting results in beef cattle fed high-concentrate diets have used corn [5] or whole-crop wheat and grass [3] silages as forage sources. Additionally, one of the manuscripts [5] indicates that the animals were implanted with Synovex-S. In feedlots, the forage used is usually of low quality [14], and implants have been forbidden in many parts of the world (Europe, for example) for many years. There is a lack of data on the use of AIA as a digestibility marker in beef cattle fed high-concentrate diets (ca. 90%) and using low-quality forages such as barley straw.
Regarding uNDF, previous attempts to validate it as an internal digestibility marker across a variety of beef diets have produced mixed results with all-forage [4,15,16,17,18,19] or high-forage [4,15,17] diets. In the very scarce papers reporting the use of uNDF as a digestibility marker in beef cattle fed high-concentrate diets, the proportion of concentrates varied between 0.75 [17] and 0.9 [4] of the dry matter (DM), and the forages used were alfalfa hay and grass hay [4] or grass silage and barn-dried grass [17]. To our knowledge, there is no literature available on the accuracy of uNDF as a digestibility marker for studies involving beef cattle fed high-concentrate diets (ca. 90%) and using low-quality forages such as barley straw.
On this basis, the objectives of the present paper were 1. to compare digestibility values obtained by total faecal collection vs. AIA or uNDF (Experiment 1) in beef cattle fed high-concentrate and barley straw diets, 2. to compare digestibility values obtained using Cr2O3 as an external marker vs. AIA or uNDF (Experiment 2) in beef cattle fed high-concentrate and barley straw diets, and 3. to compare digestibility values obtained using AIA vs. uNDF (Experiment 3) in beef cattle fed high-concentrate and barley straw diets.

2. Materials and Methods

2.1. Animals, Diets, and Experimental Procedures

The comparison of the different techniques for estimation of digestibility carried out in this paper was performed by analysing feed and faecal samples from three already published experiments. In Experiment 1 [20], digestibility was determined by total faeces collection in metabolic cages, and the internal markers AIA and uNDF were further considered in the present study. Faecal recovery of AIA and uNDF was assessed. In Experiment 2 [14], digestibility was estimated using Cr2O3 as an external marker, and then AIA and uNDF were also considered in the present paper. Finally, in Experiment 3 [21], digestibility was estimated using AIA, and then uNDF was also analysed in this study. The analysis of markers in the two latter experiments was performed on spot faeces samples; hence, recovery studies were not possible.
All three experiments used Montbéliarde crossbred male calves (314 kg average weight and 208 average days of age in Experiment 1 [20], 358 kg average weight and 251 average days of age in Experiment 2 [14], and 268 kg average weight and 190 average days of age in Experiment 3 [21]), and animal care, handling and surgical procedures (fitting of a 120 mm long, 11 mm internal diameter permanent cannula and the dorsal sac of the rumen) were approved by the Ethics Committee of the University of Zaragoza (ethical approval code PI26_21 for trials at the commercial farm Murilló Fresh Foods SL, in the Spanish Pyrenees, and ethical approval code PI57/21 for the trials carried out at the Servicio de Experimentación Animal of the University of Zaragoza). The care and management of animals were performed according to the Spanish Policy for Animal Protection RD 1201/05, which meets the EU Directive 86/609 on the protection of animals used for experimental and other scientific purposes.
Animals were offered barley straw and a concentrate, both ad libitum. The concentrate was made up with either maize or barley as the majority cereal in Experiment 1 [20] or maize as the majority cereal in Experiment 2 [14] and Experiment 3 [21]. The concentrates were formulated to have 2777 kcal ME/kg, 2814 kcal ME/kg, and 2785 kcal ME/kg in experiments 1, 2, and 3, respectively, and the intended crude protein (CP) content was 15%, 12%, and 14%. A detailed description of the ingredients used to formulate the concentrates and of the chemical composition of these latter can be found in the referenced publications [14,20,21]. The straw was offered in the long form (without processing) in Experiment 1 [20] or either in the log form or ground (6 mm sieve) and pelleted (8 mm pellet diameter) in Experiment 2 [14]. In Experiment 3 [21], pelleted barley and the concentrate were offered either in separate troughs or as a total mixed ration (10% barley and 90% concentrate). The chemical composition of the straw can also be found in the referenced papers [14,20,21].

2.2. Chemical Analysis

Analyses of feeds, feed residues, and faeces from the different experiments have already been described [14,20,21], as well as chromium [14] and AIA [21] determinations. In Experiment 1, AIA and uNDF were analysed in feeds, feed residues, and faeces; in Experiment 2, Cr was also analysed in feeds, feed residues, and faeces, but AIA and uNDF were not assessed in feed residues; in Experiment 3, neither AIA nor uNDF was determined in feed residues.
To determine uNDF, the Tilley–Terry incubation [22] was performed in a first step, followed by hydrolysis with pepsin in hydrochloric acid. Then, the neutral detergent fibre content of the residue was determined as represented in [20].

2.3. Mathematical and Statistical Methods

Faecal recoveries of AIA and uNDF were calculated in Experiment 1 following the usual procedures. Dry matter digestibility (DMD; g/kg) was estimated as follows:
D M D = 1 − M a r k e r i n t a k e M a r k e r e x c r e t e d
whereas digestibility of OM (OMD), CP (CPD), and NDF (NDFD) was calculated as follows:
O M D ,   C P D   o r   N D F D = 1 − M a r k e r i n t a k e M a r k e r e x c r e t e d × n e x c r e t e d n i n t a k e
where [Marker]intake is the concentration of AIA or uNDF in the DM consumed by the animal, [Marker]excreted is the average concentration of AIA or uNDF in the faecal samples (DM), nexcreted is the proportion of OM, CP, or NDF in the faeces, and nintake is the proportion of those fractions in intake. [Marker]intake was calculated either considering the concentration of AIA or uNDF in feed residues or dismissing these latter. 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 the first instance and comparing the results obtained considering feed residues or not.
In Experiments 2 and 3, only DMD was assessed using Cr, AIA, and uNDF (Experiment 2) or AIA and uNDF (Experiment 3).
Digestibility values obtained according to the different methods were compared using the PROC MIXED of SAS (SAS Inst. Inc., Cary, NC, USA, v 9.4). In Experiment 1, treatment (diet), method of calculation (total collection of faeces, AIA, or uNDF), digestibility balance (see [8]), and their interactions were included as fixed effects, and animal as random. As the interaction between method of calculation and balance was significant (p < 0.0001), a second analysis was performed independently for each balance. In Experiment 2 [16], the fixed effects were treatment (diet), marker (Cr, AIA, and uNDF), and their interaction, and animal was considered a random effect. Both the analysed and theoretical concentrations of Cr in the diet consumed were considered. In Experiment 3 [17], treatment (diet) and marker (AIA and uNDF) were considered as fixed effects, and animal as random.
Regressions between DMD values obtained using different methods were analysed using the PROC REG protocol of SAS. Differences were considered significant if p < 0.05.

3. Results

3.1. Experiment 1

In this experiment [20], digestibility obtained using total collection of faeces was compared to values estimated using AIA and uNDF, either considering feed residues or not.
As pointed out above, a first ANOVA was conducted considering all digestibility balances together. No treatment (corn or barley as the predominant cereal in the concentrate) effect was noted (p = 0.5660). However, there was a significant effect of the interaction between digestibility balance and method of calculation (p < 0.0001); hence, a subsequent analysis was performed for each digestibility balance independently. The results of this second analysis are summarised in Table 1.
For balances 1 and 2, there were no differences between digestibility coefficients obtained from total faeces collection or using AIA as an internal marker, irrespective of consideration of its concentration in the feed residues. However, the use of uNDF led to a substantial underestimation. Faecal recoveries of AIA varied markedly between animals and balances, with average values (±SE) of 163 ± 59.9% for balance 1 (with a range of 74–340% for the different animals), 129 ± 9.3% for balance 2 (range of 102–146%), and 22 ± 4.2% for balance 3 (range of 15–34%). Regarding uNDF, faecal recoveries were 58 ± 1.39% (54–61%) for balance 1, 65 ± 6.1% (49–76%) for balance 2, and 39 ± 4.9% (30–52%) for balance 3. During the second balance, the estimation of digestibility from uNDF, considering its concentration in the feed residues, was higher in the animals fed a concentrate with barley as the main cereal. As a result, the interaction between treatment (diet) and method of calculation of digestibility was significant (p = 0.0347). For balance 3, digestibility values obtained using AIA were extremely low, with even negative figures. uNDF-derived values were also lower in this balance than in the previous ones.
The relationships between digestibility values obtained using total faeces collection or AIA and uNDF as internal markers (either considering the concentration in the feed residues or not) are shown in Figure 1, and the corresponding regressions are shown in Table 2. In general, the coefficients of determination were low, and there was a lack of significance of the different equations. Also, the intercepts were different from zero, and the slopes different from 1 in any case. Surprisingly, the slopes of the regressions were below zero in some instances, indicating a negative relationship between digestibility values obtained using total faeces collection or the internal markers. Due to this lack of agreement between DMD values obtained using different methods, OMD, CPD, and NDFD were not included in Table 1.

3.2. Experiment 2

In this experiment [14], digestibility values estimated using Cr2O3 as an external marker were compared to those obtained using either AIA or uNDF as internal markers. Due to the lack of differences in digestibility values found in the previous experiment between considering the concentration of internal markers in feed residues or not (except for balance 3), feed residues were not analysed in this case. Regarding Cr, both the analysed concentrations in the concentrate and the theoretical ones were used to estimate digestibility.
In this trial, digestibility values estimated using AIA were higher (p = 0.0009) than those obtained using Cr (either analysed or considering the theoretical concentration in the concentrate [14]) or uNDF, with no differences between these two latter (Table 3). For the same reasons stated above, only DMD values were included in Table 3.
The regression between digestibility values obtained using the analysed vs. theoretical concentration of Cr in the concentrate [14] was significant (p < 0.001; Table 4), with a high determination coefficient (r2 = 0.9993). Additionally, the intercept was not different from 0, and the slope was not different from 1. For the rest of the relationships, the coefficients of determination were low, and there was a lack of significance of the different equations. Moreover, the intercepts were different from zero, and the slopes were different from 1 in any case. Also, the slopes of the regressions were positive in some instances but negative in others.

3.3. Experiment 3

In the third experiment [21], digestibility was estimated using AIA in spot faecal samples, and then uNDF was further analysed. As shown in Table 3, the effect of the interaction between treatment (total mixed ration vs. feeding straw and concentrate separately) and method of calculation of digestibility (using AIA or uNDF as internal markers) was significant. This interaction was caused by the differences between treatments when using uNDF as a digestibility marker but not AIA, and by the differences between markers for feeding straw and concentrate separately but not for total mixed ration. The regression between digestibility values obtained using AIA and uNDF as internal markers was not significant (uNDF = 57.0 + 0.215 AIA; p = 0.3127; r2 = 0.0678).

4. Discussion

4.1. Faecal Recovery of AIA

Several reviews [1,23,24,25] have outlined that an ideal marker must not be absorbed, which implies that faecal recovery should be complete. Despite that, some authors have reported well in-excess recoveries of AIA in beef cattle [12,26], and the reasons argued have included analytical imprecisions [2,27], soil and dust in diets or faeces [23], or inaccurate feed sampling for AIA analysis [2].
In Experiment 1 of the present study, faecal recoveries of AIA showed average values (±SE) of 163 ± 59.9% for balance 1 (with a range of 74–340% for the different animals), 129 ± 9.3% for balance 2 (range of 102–146%), and 22 ± 4.2% for balance 3 (range of 15–34%). Trying to reduce uncertainties, the reliability of the technique employed to analyse AIA [28] was first checked. This technique includes two methods: A, for organic raw ingredients of a compound feed, and most compound feeds, and B, for raw ingredients, mineral premixes, and compound feeds with an AIA content estimated by method A higher than 1%. Method A was first applied, and the average concentrations obtained were as follows: 1. for balance 1, 24.21 g AIA/kg DM in the straw, 1.59 g AIA/kg DM in the concentrate with a higher proportion of maize, 3.55 g AIA/kg DM in the concentrate with a higher proportion of barley, and 16.63, 25.38, 84.11, and 15.98 g AIA/kg DM in the faeces of the four animals employed; 2. for balance 2, the values were, respectively, 11.32, 2.31, and 3.96, and 20.10, 16.29, 22.40, and 23.04 g AIA/kg DM; and 3. for balance 3, the results were 41.04, 2.18, and 3.65, and 17.45, 14.25, 14.87, and 10.56 g AIA/kg DM. When the concentrations were higher than 1%, the samples were re-analysed using protocol B [28]. However, differences between methods A and B were less than 10%, on average, and estimations of digestibility were not substantially improved (less than 5% change). For this reason, the values obtained with method A [28] were maintained. The high content of AIA in the faeces of one of the animals from balance 1 (84.11 g/kg DM) was responsible for the recovery of 340% and might have been due to contamination of the faeces with soil. It must be pointed out that in Experiment 1 [21], metabolism cages were placed outside the barns in a commercial farm, and that rain was present during some days of balance 1, sweeping along soil from an adjacent hillside. Our metabolism cages do not avoid part of the faeces falling on the ground, which has to be manually collected to account for total collection. Contamination of these faeces with soil is not likely to dramatically increase faecal output, but, in contrast, it is likely to increase the concentration of AIA in the collected faeces because much of the AIA is silica [5,27]. It must be considered that total faeces were collected daily from each animal, and a 5% aliquot was taken, frozen at −20 °C, pooled by balance, and freeze-dried. This way, we ensured that the final sample of faeces was collected in different places, thus minimising the impact of collecting just the material in close contact with the soil. The extremely low values of AIA recovery found during balance 3 could have been due to a contamination of the straw and/or the concentrate with soil. The ash content of straw in balances 1, 2, and 3 were, respectively, 54.7 g/kg DM, 66.9 g/kg DM, and 91.6 g/kg DM. In addition, the AIA content was 24.2, 11.3, and 41.0 g/kg DM. As stated above, an increase in the concentration of AIA in the straw and/or in the concentrate due to contamination with soil or dust is likely to dramatically decrease faecal recovery of this internal marker.

4.2. Faecal Recovery of uNDF

The incomplete faecal recoveries of uNDF obtained in the present study have also been observed across the literature [15], and the reasons argued include lack of uniform particle size among fermentation substrates [16,29], forage quality [16], or faecal sampling errors or analytical errors associated with the uNDF method [4,19,30]. The concentrates in Experiment 1 were offered in the form of a 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 study, as the diets included ca. 90% concentrate, with less variability across particle sizes than forages. Moreover, the expected low particle size of the concentrates could have produced more errors when applying the method for estimation of uNDF [22]. In our opinion, an effort should be made to develop techniques to obtain uNDF of concentrates with a high degree of accuracy. In this context, faecal recovery of uNDF in animals fed mixed diets seems to be more dependent on forage type rather than on the forage-to-concentrate ratio (60:40 or 40:60) [31].

4.3. Digestibility Method Evaluation

Even though total faeces collection is considered the gold standard for digestibility studies, it has been pointed out that the faecal sampling method may influence the results obtained. In our study, collection was performed once a day; however, watching the animals constantly and gathering faeces immediately after bowel movement has been shown to reduce contamination or partial loss of the material [31]. This practice could have overcome the limitations stated in the previous section due to the design of our metabolism cages, and we can hypothesise that more accurate faecal recoveries of AIA and uNDF could have been obtained.

4.3.1. Experiment 1

In Experiment 1, and dismissing the results obtained during balance 3 due to the very low recoveries of both AIA and uNDF, there were no differences between digestibility coefficients obtained from total faeces collection or using AIA as an internal marker, irrespective of consideration of its concentration in the feed residues (Table 1). In other words, it seems that, when using AIA as an internal marker to estimate digestibility, there is no need to analyse its concentration in feed residues.
Even though there were no differences between average values obtained by total faeces collection or using AIA, Figure 1 and Table 2 show a lack of relationship between individual digestibility values obtained by both methods. Sales and Janssens [2] reported that of 45 studies where the AIA method was compared to the total collection method to determine digestibility of feeds in different animal species, 26 showed similar results, 9 showed an underestimation by the AIA method, and 10 showed an overestimation. In this context, not only underestimation of nutrient digestibility using AIA in lactating Holstein cows [32] but also a strong relationship between AIA and total faeces collection have been reported [3,33]. Sales and Janssens [2] attributed the failure of AIA as a marker mostly to analytical errors, especially in feeds with low natural AIA content [5], and concluded that AIA presents a reliable marker with several advantages that could be successfully utilised to determine faecal digestibility in animal species under certain circumstances and with the application of some precautions. The results of the present study, however, indicate that the use of AIA as an internal marker for digestibility studies in cattle fed high-concentrate diets seems to produce average values comparable to those obtained by total faeces collection, provided there is no contamination of feedstuffs and/or faeces with dust and/or soil, but individual values are not accurately obtained. Due to the accumulation of uncertainties, our recommendation would be not to use AIA as an internal marker for digestibility studies in intensively reared cattle when individual accurate values are needed.
The low faecal recoveries found for uNDF, for the reasons stated in Section 4.1, are responsible for the low digestibility values obtained using this marker in Experiment 1. As for AIA, there was a lack of relationship between individual digestibility values obtained by total faeces collection or uNDF (Figure 1). Contrasting results have been reported in the literature, where good agreement between digestibility values using total faeces collection or uNDF has been found in lactating Holstein cows with faeces collection carried out every 2 h on a 24 h cycle [31,32]. The authors of the latter paper [32] recommend a sampling frequency of at least six events to obtain accurate estimates. As for AIA, and for the same reasons, this internal marker is also not recommended for digestibility studies in cattle fed high-concentrate diets.

4.3.2. Experiment 2

There was no total faeces collection in Experiment 2, but the digestibility values obtained using Cr2O3 as an external marker were considered reliable. Many papers have used chromic oxide as an external digestibility marker [34,35,36,37,38,39], and concerns about incomplete or excessive recovery [34] can be easily overcome with an appropriate analysis [40]. Preoccupation about health and environmental risks [35,41] can be reduced by using individual protection of laboratory workers and appropriate disposal of waste as dangerous residues [42], and large diurnal variation in faecal concentrations can be dealt with by collecting enough faeces samples throughout the day to provide an average sample in which the marker concentration is representative of that over the entire day [34,42].
In this experiment, there were no differences between digestibility values estimated using either analysed Cr or considering its theoretical concentration in the concentrate (Table 3). Also, the regression between both digestibility values was significant (Table 4), and with a high determination coefficient, the intercept was not different from 0, and the slope was not different from 1. This implies that analysis of chromium in the labelled concentrate would not be needed if correct homogenisation of the marker (as was our case) can be assured by any means other than chemical determination in different samples.
In contrast to the results found in Experiment 1, in Experiment 2, digestibility values estimated using AIA were statistically higher than those obtained using Cr, with no differences between the latter and uNDF (Table 3). It could be argued that, even though Cr usually gives good and accurate estimations of digestibility, this might not have been the case in this trial. However, uncertainties pointed out above about faecal recovery of AIA and uNDF may also be involved. Other authors [43] have indicated that AIA is not an acceptable internal marker for digestibility studies in cattle fed diets common to feedlot systems and that, when using uNDF, at least two spot samplings should be collected. This was the case in Experiment 2 [14], and this fact might have helped the lack of significant differences between Cr2O3 and uNDF-derived digestibility values. Also, as in Experiment 1, the regressions between dry matter digestibility values obtained using Cr2O3 and internal markers were not significant (Table 4) and had low coefficients of determination.

4.3.3. Experiment 3

In Experiment 3, AIA and uNDF markers gave different values of digestibility (lower for uNDF, as in Experiment 1) when the ration was given as TMR [21] (Table 3) but not when the straw and the concentrate were administered in separate troughs. Other authors [44] have not only claimed that uNDF appears to be a more reliable digestibility marker than AIA in terms of detecting dietary differences in apparent digestibility of some nutrients but also have pointed out that care must be taken when significant diet × digestibility marker interactions are observed when estimating total-tract digestibility using intrinsic markers. In this respect, the accuracy of digestibility values obtained using uNDF as an internal marker seems to be due to its concentration in the diet [44].
The inconsistent performance of AIA and uNDF across experiments 1–3 weakened their practical application. Feed types and housing systems, among other factors, could explain inconsistencies and should be considered in future research in this field.

5. Conclusions

Faecal recovery of AIA and uNDF is highly variable and depends on many factors that cannot be easily fixed in nutrition experiments. As a result, digestibility estimated using those internal markers may also be greatly variable and inaccurate.
The use of the above-mentioned internal markers cannot be recommended to accurately estimate digestibility in beef cattle fed high-concentrate diets, with the external marker Cr2O3 preferred under controlled sampling. AIA could be used, in any case, only for mean estimates in clean indoor settings.

Author Contributions

Conceptualisation, A.d.V.; methodology, A.A. and A.d.V.; validation, A.d.V.; formal analysis, A.d.V.; investigation, A.A. and A.d.V.; resources, A.d.V.; data curation, A.A. and A.d.V.; writing—original draft preparation, A.A.; writing—review and editing, A.d.V.; visualisation, A.d.V.; supervision, A.d.V.; project administration, A.d.V.; funding acquisition, A.d.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the ‘Centro para el Desarrollo Tecnológico Industrial’ (Spanish Ministry for Science and Innovation) (grant number: IDI-20200342).

Institutional Review Board Statement

This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the University of Zaragoza (protocol code PI26_21, approved on 22 March 2021, and protocol PI57_21, approved on 17 November 2021).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

Authors would like to acknowledge the use of Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza (Servicio de Experimentación Animal and Servicio de Análisis Químicos), for its support.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of this study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Owens, F.N.; Hanson, C.F. External and internal markers for appraising site and extent of digestion in ruminants. J. Dairy Sci. 1992, 75, 2605–2617. [Google Scholar] [CrossRef] [PubMed]
  2. Sales, J.; Janssens, G.P.J. Acid-insoluble ash as a marker in digestibility studies: A review. J. Anim. Feed Sci. 2003, 12, 383–401. [Google Scholar] [CrossRef]
  3. Mc Geough, E.J.; O’Kiely, P.O.; Kenny, D.A. A note on the evaluation of the acid-insoluble ash technique as a method for determining apparent diet digestibility in beef cattle. Irish J. Agric. Food Res. 2010, 49, 159–164. [Google Scholar]
  4. Peters, J.F.; Swift, M.L.; Penner, G.B.; Lardner, H.A.; McAllister, T.A.; Ribeiro, G.O. Comparison of two internal markers and near-infrared spectroscopy (NIRS) for predicting nutrient digestibility in beef cattle offered diets varying in forage quantity and quality. Can. J. Anim. Sci. 2025, 105, 1–12. [Google Scholar] [CrossRef]
  5. Thonney, M.L.; Palhof, B.A.; DeCarlo, M.R.; Ross, D.A.; Firth, N.L.; Quaas, R.L.; Perosio, D.J.; Duhaime, D.J.; Rollins, S.R.; Nour, A.Y.M. Sources of variation of dry matter digestibility measured by the acid insoluble ash marker. J. Dairy Sci. 1985, 68, 661–668. [Google Scholar] [CrossRef]
  6. Paloheimo, L.; Mäkelä, A. The rate of passage of food in the digestive tract of ruminants. Maatal. Aikakauskirje 1952, 24, 165–169. [Google Scholar] [CrossRef]
  7. Reid, J.T.; Woolfolk, P.G.; Richards, C.R.; Kaufmann, R.W.; Loosli, J.K.; Turk, K.L.; Miller, J.I.; Blaser, R.E. A new indicator method for the determination of digestibility and consumption of forages by ruminants. J. Dairy Sci. 1950, 33, 60–71. [Google Scholar] [CrossRef]
  8. Penning, P.D.; Johnson, R.H. The use of internal markers to estimate herbage digestibility and intake. 1. Potentially indigestible cellulose and acid insoluble ash. J. Agric. Sci. 1983, 100, 127–131. [Google Scholar] [CrossRef]
  9. Valiente, O.L.; Delgado, P.; de Vega, A.; Guada, J.A. Validation of the n-alkane technique to estimate intake, digestibility, and diet composition in sheep consuming mixed grain:roughage diets. Aust. J. Agric. Res. 2003, 54, 693–702. [Google Scholar] [CrossRef]
  10. Uden, P.; Colucci, P.E.; Van Soest, P.J. Investigation of chromium, cerium and cobalt as markers in digesta. Rate of passage studies. J. Sci. Food Agric. 1980, 31, 625–632. [Google Scholar] [CrossRef]
  11. Siddons, R.C.; Paradine, J.; Beever, D.E.; Cornell, P.R. Ytterbium acetate as a particulate-phase digesta-flow marker. Br. J. Nutr. 1985, 54, 509–519. [Google Scholar] [CrossRef] [PubMed]
  12. Sunvold, G.D.; Cochran, R.C. Technical note: Evaluation of acid detergent lignin, alkaline peroxide lignin, acid insoluble ash, and indigestible acid detergent fiber as internal markers for prediction of alfalfa, bromegrass, and prairie hay digestibility by beef steers. J. Anim. Sci. 1991, 69, 4951–4955. [Google Scholar] [CrossRef] [PubMed]
  13. Ramos, B.; Banchero, G.; La Manna, A.; Andrighetto-Canozzi, M.E.; Fernández, E.; Clariget, J. Applicability of an automated supplement feeder to dose titanium dioxide to estimate forage intake, fecal output, and diet digestibility of beef cattle. Appl. Anim. Sci. 2024, 40, 132–141. [Google Scholar] [CrossRef]
  14. Arbaoui, A.; Gonzalo, G.; Belanche, A.; de Vega, A. Can feedlot cattle increase productivity and decrease methane emissions by lowering the straw particle size? Anim. Feed Sci. Technol. 2025, 323, 116282. [Google Scholar] [CrossRef]
  15. Cochran, R.C.; Adams, D.C.; Wallace, J.D.; Galyean, M.L. Predicting digestibility of different diets with internal markers: Evaluation of four potential markers. J. Anim. Sci. 1986, 63, 1476–1483. [Google Scholar] [CrossRef]
  16. Lippke, H.; Ellis, W.C.; Jacobs, B.F. Recovery of indigestible fiber from feces of sheep and cattle on forage diets. J. Dairy Sci. 1986, 69, 403–412. [Google Scholar] [CrossRef]
  17. Huhtanen, P.; Kaustell, K.; Jaakkola, S. The use of internal markers to predict total digestibility and duodenal flow of nutrients in cattle given six different diets. Anim. Feed Sci. Technol. 1994, 48, 211–227. [Google Scholar] [CrossRef]
  18. Rønn, M.; Bach Knudsen, K.E.; Kristensen, N.B.; Weisbjerg, M.R. Can lignin and monomer composition of fibre describe the variation in iNDF in forages? Anim. Feed Sci. Technol. 2022, 284, 115157. [Google Scholar] [CrossRef]
  19. Jonker, A.; Della Rosa, M.M. Comparing three filter-bag types for accuracy and precision of in sacco undigestible neutral detergent fibre determination of various dicotyledon forages. Anim. Prod. Sci. 2024, 64, AN24281. [Google Scholar] [CrossRef]
  20. Arbaoui, A.; de Vega, A. Does replacing maize with barley affect the animal performance and rumen fermentation, including methane production, of beef cattle fed high-concentrate diets on-farm? Animals 2023, 13, 3016. [Google Scholar] [CrossRef]
  21. Arbaoui, A.; Gonzalo, G.; Belanche, A.; de Vega, A. Unmixed Diet Versus Total Mixed Ration in Beef Cattle Fed High-Concentrate Diets: Effects on Methane Emissions, Animal Performance, and Rumen Fermentation. Animals 2025, 15, 723. [Google Scholar] [CrossRef] [PubMed]
  22. Tilley, J.M.A.; Terry, R.A. A two-stage technique for the in vitro digestion of forage crops. Grass Forage Sci. 1963, 18, 104–111. [Google Scholar] [CrossRef]
  23. Marais, J.P. Use of markers. In Farm Animal Metabolism and Nutrition; Mello, J.P.F., Ed.; CABI International: Wallingford, UK, 2000; pp. 255–277. [Google Scholar]
  24. Faichney, G.J. The use of markers to partition digestion within the gastrointestinal tract of ruminants. In Digestion and Metabolism in the Ruminant; McDonald, I.W., Warner, A.C.I., Eds.; The University of New England Publishing Unit: Armidale, Australia, 1975; pp. 277–291. [Google Scholar]
  25. Fahey, G.C.; Jung, H.G. Lignin as a marker in digestion studies: A review. J. Anim. Sci. 1983, 57, 220–225. [Google Scholar] [CrossRef]
  26. Wilson, R.K.; Winter, K.A. An evaluation of acid-insoluble ash as a predictor of feed digestibility in ruminants. Irish J. Agric. Res. 1984, 23, 97–98. [Google Scholar]
  27. Van Keulen, J.; Young, B.A. Evaluation of acid-insoluble ash as a natural marker in ruminant digestibility studies. J. Anim. Sci. 1977, 44, 282–287. [Google Scholar] [CrossRef]
  28. Official Journal of the European Union. Commission Implementing Regulation (EU) 2024/771 of 29 February 2024 Amending Regulation (EC) No 152/2009 Laying Down the Methods of Sampling and Analysis for the Official Control of Feed. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=celex:32024R0771 (accessed on 23 May 2024).
  29. Wilkins, R.J.; Minson, D.J. The effects of grinding, supplementation and incubation period on cellulose digestibility in vitro and its relationship with cellulose and organic matter digestibility in vivo. J. Agric. Sci 1970, 74, 445–451. [Google Scholar] [CrossRef]
  30. Raffrenato, E.; Ross, D.A.; Van Amburgh, M.E. Development of an in vitro method to determine rumen undigested aNDFom for use in feed evaluation. J. Dairy Sci. 2018, 101, 9888–9900. [Google Scholar] [CrossRef]
  31. Velásquez, A.V.; Oliveira, C.A.; Martins, C.M.M.R.; Balieiro, J.C.C.; Silva, L.F.P.; Fukushima, R.S.; Sousa, D.O. Diet, marker and fecal sampling method interactions with internal and external marker pairs when estimating dry matter intake in beef cattle. Livest. Sci. 2021, 253, 104730. [Google Scholar] [CrossRef]
  32. Morris, D.L.; Rebelo, L.R.; Dieter, P.A.; Lee, C. Validating intrinsic markers and optimizing spot sampling frequency to estimate fecal outputs. J. Dairy Sci. 2018, 101, 7980–7989. [Google Scholar] [CrossRef]
  33. Nousiainen, J.; Rinne, M.; Huhtanen, P. A meta-analysis of feed digestion in dairy cows. The effects of forage and concentrate factors on total diet digestibility. J. Dairy Sci. 2009, 92, 5019–5030. [Google Scholar] [CrossRef]
  34. Titgemeyer, E.C. Design and interpretation of nutrient digestion studies. J. Anim. Sci. 1997, 75, 2235–2247. [Google Scholar] [CrossRef] [PubMed]
  35. Myers, W.D.; Ludden, P.A.; Nayigihugu, V.; Hess, B.W. Excretion patterns of titanium dioxide and chromic oxide in duodenal digesta and feces of ewes. Small Rumin. Res. 2006, 63, 135–141. [Google Scholar] [CrossRef]
  36. Borucki Castro, S.I.; Lapierre, H.; Phillip, L.E.; Jardon, P.W.; Berthiaume, R. Towards non-invasive methods to determine the effect of treatment of soya-bean meal on lysine availability in dairy cows. Animal 2008, 2, 224–234. [Google Scholar] [CrossRef] [PubMed]
  37. Amorocho, A.K.; Jenkins, T.C.; Staples, C.R. Evaluation of catfish oil as a feedstuff for lactating Holstein cows. J. Dairy Sci. 2009, 92, 5178–5188. [Google Scholar] [CrossRef]
  38. Delagarde, R.; Perez-Ramirez, E.; Peyraud, J.L. Ytterbium oxide has the same accuracy as chromic oxide for estimating variations of faecal dry matter output in dairy cows fed a total mixed ration at two feeding levels. Anim. Feed Sci. Technol. 2010, 161, 121–131. [Google Scholar] [CrossRef]
  39. Schäfers, S.; Bulang, M.; Meyer, U.; Lindwedel, A.; Hüther, L.; Dänicke, S. Suitability of n-alkanes and chromium (III) oxide as digestibility markers in calves at the end of the milk feeding period supplemented with a prebiotic. Anim. Nutr. 2018, 4, 84–89. [Google Scholar] [CrossRef]
  40. Vicente, F.; Sarraseca, A.; deVega, A.; Guada, J.A. Performance of several Cr and Yb analytical techniques applied to samples of different biological origin (digesta or faeces). J. Sci. Food Agric. 2004, 84, 2035–2040. [Google Scholar] [CrossRef]
  41. Titgemeyer, E.C.; Armendariz, C.K.; Bindel, D.J.; Greenwood, R.H.; Loest, C.A. Evaluation of titanium dioxide as a digestibility marker for cattle. J. Anim. Sci. 2001, 79, 1059–1063. [Google Scholar] [CrossRef]
  42. Al Alami, A.; Gimeno, A.; de Vega, A.; Fondevila, M.; Castrillo, C. Effects of Cr2O3 labelling dose, and of faeces sampling schedule, on faecal Cr concentration and on digestibility estimation in cattle fed high-concentrate diets. Livest. Sci. 2014, 168, 53–59. [Google Scholar] [CrossRef]
  43. Beck, M.R.; Proctor, J.A.; Smith, J.K.; Gouvêa, V.N.; Kasuske, Z.; Foote, A.P.; Gunter, S.A.; Beck, P.A. Assessing different sampling regimens for estimating dietary characteristics using internal markers. Appl. Anim. Sci. 2023, 39, 411–422. [Google Scholar] [CrossRef]
  44. Lee, C.; Hristov, A.N. Short communication: Evaluation of acid-insoluble ash and indigestible neutral detergent fiber as total-tract digestibility markers in dairy cows fed corn silage-based diets. J. Dairy Sci. 2013, 96, 5295–5299. [Google Scholar] [CrossRef]
Agriculture 15 01485 g001
Figure 1. Relationships between digestibility values obtained using total faeces collection (FC) or AIA and uNDF as internal markers (either considering the concentration in the feed residues or not) in feedlot beef cattle consuming different concentrates and barley straw during three different digestibility balances (■, □, and ●) [20].
Figure 1. Relationships between digestibility values obtained using total faeces collection (FC) or AIA and uNDF as internal markers (either considering the concentration in the feed residues or not) in feedlot beef cattle consuming different concentrates and barley straw during three different digestibility balances (■, □, and ●) [20].
Agriculture 15 01485 g001
Table 1. Digestibility coefficients (g/100 g) of dry matter in feedlot beef cattle consuming different concentrates and barley straw during three different digestibility balances [20].
Table 1. Digestibility coefficients (g/100 g) of dry matter in feedlot beef cattle consuming different concentrates and barley straw during three different digestibility balances [20].
p-Value
FCAIA-Feed ResiduesAIA-No Feed ResiduesuNDF-Feed ResiduesuNDF-No Feed ResiduesSEMTMeT × Me
Balance 1 73.95 a80.93 a72.08 a59.27 b52.78 b4.7460.12810.00090.2109
Balance 2M75.50 a82.46 a78.81 a57.50 bA57.19 b3.3490.8913<0.00010.0347
B72.75 a78.73 a71.72 ab65.22 bB64.08 b
Balance 3 72.70 a−13.65 c1.37 c38.36 b38.32 b12.1990.23140.00030.9963
FC: digestibility estimated by total faeces collection; AIA: digestibility estimated using acid-insoluble ashes as an internal marker; uNDF: digestibility estimated using undigestible neutral detergent fibre as an internal marker; feed residues: digestibility estimated taking into account the concentration of markers in the feed residues; no feed residues: digestibility estimated dismissing the concentration of markers in the feed residues; SEM: standard error of the mean; T: treatment (maize—M or barley—B as the predominant cereal in the concentrate); Me: method of calculation (FC, AIA, or uNDF, with or without taking into consideration the concentration of markers in the feed residues); a, b: different letters within a row indicate differences between methods of calculation (within a treatment for balance 2) at p < 0.05; and A, B: different letters within a column indicate differences between treatments for balance 2 at p < 0.05.
Table 2. Regressions between dry matter digestibility values obtained using total faeces collection (FC –x-) or internal markers (y) in feedlot beef cattle consuming different concentrates and barley straw during three different digestibility balances [20].
Table 2. Regressions between dry matter digestibility values obtained using total faeces collection (FC –x-) or internal markers (y) in feedlot beef cattle consuming different concentrates and barley straw during three different digestibility balances [20].
Equationr2p-Value
FC vs. AIA-feed residuesy = −283 + 4.52 x 0.04110.5273
 Balance 1y = 509 − 5.79 x0.75670.1301
 Balance 2y = −5.16 + 1.16 x0.41950.3523
 Balance 3y = 164 − 2.45 x0.09060.6989
FC vs. AIA-no feed residuesy = −222 + 3.70 x0.04660.5003
 Balance 1y = 727 − 8.86 x0.90890.0466
 Balance 2y = −92.8 + 2.27 x0.59450.2290
 Balance 3y = 84.7 − 1.15 x0.04870.7793
FC vs. uNDF-feed residuesy = −27.2 + 1.09 x0.03800.5439
 Balance 1y = −43.4 + 1.39 x0.38810.3771
 Balance 2y = 268 − 2.79 x0.90440.0490
 Balance 3y = 39.0 − 0.009 x0.00000.9968
FC vs. uNDF-no feed residuesy = −18.7 + 0.941 x0.03070.5862
 Balance 1y =118 − 0.884 x0.05410.7675
 Balance 2y = 243 − 2.46 x0.99650.0017
 Balance 3y = 9.18 + 0.401 x0.01460.8790
AIA: digestibility estimated using acid insoluble ashes as an internal marker; uNDF: digestibility estimated using undigestible neutral detergent fibre as an internal marker; feed residues: digestibility estimated considering the concentration of markers in the feed residues; no feed residues: digestibility estimated dismissing the concentration of markers in the feed residues.
Table 3. Digestibility coefficients (g/100 g) of dry matter in feedlot beef cattle consuming a concentrate and barley straw in the long or ground and pelleted form (Experiment 2 [14]), or using a total mixed ration compared to distributing the concentrate and the straw separately (Experiment 3 [21]).
Table 3. Digestibility coefficients (g/100 g) of dry matter in feedlot beef cattle consuming a concentrate and barley straw in the long or ground and pelleted form (Experiment 2 [14]), or using a total mixed ration compared to distributing the concentrate and the straw separately (Experiment 3 [21]).
p-Value
Cr AnalysedCr TheoreticalAIAuNDFSEMTMeT × Me
Experiment 2 70.07 b66.11b79.23 a70.79 b3.1790.19860.00090.3477
Experiment 3PC 70.24 a54.58 bB4.0500.02610.00470.0220
TMR 70.0268.20 A
Cr analysed: digestibility estimated using the analysed concentration of Cr in the concentrate [14]; Cr theoretical: digestibility estimated using the theoretical concentration of Cr in the concentrate [14]; AIA: digestibility estimated using acid insoluble ashes as an internal marker; uNDF: digestibility estimated using undigestible neutral detergent fibre as an internal marker; SEM: standard error of the mean; T: treatment (long vs. ground and pelleted straw for Experiment 2, and total mixed ration vs. distributing the concentrate and the straw separately for Experiment 3); Me: method of calculation (Cr analysed or theoretical, AIA, or uNDF); a, b: different letters within a row indicate differences between methods of calculation at p < 0.05; and A, B: different letters within a column indicate differences between treatments for Experiment 3 at p < 0.05.
Table 4. Regressions between dry matter digestibility values obtained using Cr2O3 and internal markers in feedlot beef cattle fed a concentrate and barley straw in the long or ground and pelleted form [14].
Table 4. Regressions between dry matter digestibility values obtained using Cr2O3 and internal markers in feedlot beef cattle fed a concentrate and barley straw in the long or ground and pelleted form [14].
Equationr2p-Value
Analysed (x) vs. theoretical (y) Cr in the concentratey = 11.7 + 0.883 x 0.9993<0.0001
Analysed Cr (x) vs. AIA (y)y = 82.8 − 0.160 x0.24030.0751
Analysed Cr (x) vs. uNDF (y)y = 95.8 − 0.372 x0.27710.0646
AIA (x) vs. uNDF (y)y = 50.2 + 0.451 x0.06380.3453
AIA: digestibility estimated using acid insoluble ashes as an internal marker; uNDF: digestibility estimated using undigestible neutral detergent fibre as an internal marker.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Arbaoui, A.; de Vega, A. Reliability of Acid-Insoluble Ashes and Undigestible Neutral Detergent Fibre as Internal Markers for Estimation of Digestibility in Beef Cattle Fed High-Concentrate Diets. Agriculture 2025, 15, 1485. https://doi.org/10.3390/agriculture15141485

AMA Style

Arbaoui A, de Vega A. Reliability of Acid-Insoluble Ashes and Undigestible Neutral Detergent Fibre as Internal Markers for Estimation of Digestibility in Beef Cattle Fed High-Concentrate Diets. Agriculture. 2025; 15(14):1485. https://doi.org/10.3390/agriculture15141485

Chicago/Turabian Style

Arbaoui, Amira, and Antonio de Vega. 2025. "Reliability of Acid-Insoluble Ashes and Undigestible Neutral Detergent Fibre as Internal Markers for Estimation of Digestibility in Beef Cattle Fed High-Concentrate Diets" Agriculture 15, no. 14: 1485. https://doi.org/10.3390/agriculture15141485

APA Style

Arbaoui, A., & de Vega, A. (2025). Reliability of Acid-Insoluble Ashes and Undigestible Neutral Detergent Fibre as Internal Markers for Estimation of Digestibility in Beef Cattle Fed High-Concentrate Diets. Agriculture, 15(14), 1485. https://doi.org/10.3390/agriculture15141485

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop