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Agricultural and Biological Sciences » "Herbivores", book edited by Vonnie D. C. Shields, ISBN 978-953-51-2988-2, Print ISBN 978-953-51-2987-5, Published: March 8, 2017 under CC BY 3.0 license. © The Author(s).

Chapter 3

Utilization of Biomarkers to Study the Grazing Behavior of Herbivore Species

By Luis Miguel Mendes Ferreira, Rafael Celaya, Ana Sofia Santos, Koldo Osoro and Miguel Rodrigues
DOI: 10.5772/67345

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Schematic diagram of factors affecting diet selection of herbivores and its effects on animal production and biodiversity in a grazing system.
Figure 1. Schematic diagram of factors affecting diet selection of herbivores and its effects on animal production and biodiversity in a grazing system.
Different herbivore species grazing heathland vegetation communities with adjacent areas of improved pasture of the north of Spain.
Figure 2. Different herbivore species grazing heathland vegetation communities with adjacent areas of improved pasture of the north of Spain.
Effect of carbon-chain length on the faecal recovery (%) of alkanes [94], long-chain fatty acids (LFCA) and long-chain alcohols (LCOH) observed in ruminant species.
Figure 3. Effect of carbon-chain length on the faecal recovery (%) of alkanes [94], long-chain fatty acids (LFCA) and long-chain alcohols (LCOH) observed in ruminant species.
Effect of carbon-chain length on the faecal recovery (%) of alkanes [109–111], long-chain fatty acids (LFCA) and long-chain alcohols (LCOH) observed in non-ruminant species.
Figure 4. Effect of carbon-chain length on the faecal recovery (%) of alkanes [109–111], long-chain fatty acids (LFCA) and long-chain alcohols (LCOH) observed in non-ruminant species.

Utilization of Biomarkers to Study the Grazing Behavior of Herbivore Species

Luis Miguel Mendes Ferreira1, Rafael Celaya2, Ana Sofia Santos3, Koldo Osoro2 and Miguel Rodrigues1
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Knowledge on diet selection of different herbivore species under each specific vegetation community is essential to develop and apply appropriate management decisions for each grazing system in order to, simultaneously, have a more efficient and sustainable utilization of pasture resources and the best animal performance level. In this chapter, traditional and more recent methodologies that can be used for studying diet selection of both domestic and wild herbivores are briefly presented, identifying the main advantages and limitations of their use. Particular emphasis is given to the utilization of epicuticular compounds, namely alkanes, long-chain fatty acids and long-chain alcohols, as faecal markers. The validation of their use is presented taking into account studies performed with different animal species under controlled conditions. The main advantages and shortcomings for their application to field studies with grazing animals are highlighted. Data indicate that the combination of these epicuticular compounds seems promising to overcome the enumerated constraints, allowing its application to more complex vegetation communities.

Keywords: diet selection, faecal markers, ruminant species

1. Introduction

The success of the strategies for the management of herbivores grazing on different plant communities, driven by production or environmental goals, requires the understanding of the processes involved in plant-herbivore interactions and their consequences for both plants and herbivores [1]. The plant-herbivore interaction is mutual and dynamic. The structure, composition, productivity, nutritive value and distribution of the different plant communities determine the intake and nutritional status of the animals [2, 3]. In turn, the herbivores, through grazing, trampling, defecation, urination, etc., affect the dynamics of the vegetation community [4, 5]. These interrelationships are specific for each herbivore species and each vegetation type and are still poorly understood, leading to the use of less appropriate management strategies for agricultural and other land use objectives [4, 6]. The type (cutting, grazing or a mixed system) and intensity level of management will have a determinant role on the evolution of the habitat and on the biodiversity, being extremely important on the maintenance of species balance, maturity and nutritive value in plant communities, relating the timing and severity of defoliation in relation to patterns of plant growth and maturity, and proposed objectives (animal performance, biodiversity, sustainability, etc.).

The understanding of the grazing behaviour, especially diet selection, of different animal species under diverse conditions is essential to develop an appropriate grazing strategy for each specific situation in order to have a more efficient and sustainable utilization of the existing vegetation ( Figure 1 ). The different dietary choices between plant species and plant parts in a specific vegetation community offered to the grazing animals are the main mechanism through which herbivores could increase sward heterogeneity [3, 7]. The diet selected by animals is constrained by temporal and spatial changes in the sward structure, plant defence mechanisms, food availability, plant phenology and animal factors [6, 7], and it differs between animal species [6, 8, 9] and also between breeds of cattle [10], sheep [11] and goats [12].


Figure 1.

Schematic diagram of factors affecting diet selection of herbivores and its effects on animal production and biodiversity in a grazing system.

Generally, ruminant species are classified into three feeding types according to morphological and physiological adaptations of the digestive system [6, 1315]: concentrate selectors (browsers), intermediate feeders and grass-roughage eaters (grazers). Based on this classification, it has been assumed that ruminant grazers, with greater body weight, achieve a higher extraction of nutrients from the diet consumed than browsers with low body weight [16]. According to Pérez-Barbería et al. [15] and Udén and van Soest [17], this is due to a higher extent of digestion of fibre by means of higher food retention in the rumen, larger stomach capacity, higher degree of stomach compartmentalization and smaller openings between the rumen and omasum. In contrast, small ruminants would compensate this lower digestion capacity by selecting high-quality plant parts such as fruits, pods, young shoots and leaves.

Previous studies [9, 18], carried out in heathland vegetation communities with adjacent areas of improved pasture (Lolium perenne and Trifolium repens) ( Figure 2 ) across the grazing season (May–December), indicate an almost total preference for herbaceous species by cattle, contrasting with the higher preference for the woody species (Erica spp., Calluna vulgaris and Ulex gallii) revealed by goats. By contrast, horses and sheep showed an intermediate behaviour, increasing the selection of the woody species through the grazing season as a result of the decrease in the availability of the preferred herbaceous species [18]. These differences in the grazing behaviour were reflected in animal performance [19]. This distinct behaviour and variable responses of different animal species allow alternative strategies to develop viable systems aiming to achieve production and biodiversity outcomes. Therefore, the evaluation of diet composition of grazing animals is important for the achievement of sustainable management and production systems for each vegetation community.


Figure 2.

Different herbivore species grazing heathland vegetation communities with adjacent areas of improved pasture of the north of Spain.

In this review, we aim to describe several methodologies that are available to assess plant-animal interactions, with particular relevance to the utilization of epicuticular compounds. The main advantages and limitations of each method are also explored, comparing the accuracy of diet composition estimates.

2. Techniques used to estimate diet composition in herbivores

Traditional techniques used to estimate diet composition of grazing animals are based either on measurements on the plant biomass (the utilization techniques) or on animal-based measurements [20], namely the direct observation of the grazing animal and the microhistological examination of plant fragments in different samples. However, all these techniques have important limitations associated with the measurement processes themselves, as the normal foraging behaviour may be compromised, and with the accuracy of the estimations [21].

Direct observation of the number of bites and the feeding times spent by the grazing animals on different plant communities is frequently used. The simplicity and minor equipment requirements are pointed out as advantages of this approach. However, as stated by Holechek et al. [20], it is extremely difficult to identify the plant species being consumed, especially when there is no spatial separation between plant species, and to convert the grazing times or number of bites to an accurate estimate of the amount of the plant consumed [22], besides being a time-consuming approach that is very difficult to accomplish during nocturnal periods.

The microhistological procedures rely on the visual identification of epidermal cuticular fragments in samples of oesophageal extrusa, in a gut compartment or in faeces [20, 21]. Diet composition is expressed in terms of the proportion of identifiable fragments coming from each plant species. Although microhistological approaches can be valuable to confirm the presence or the absence of a particular plant species or plant part in the diet [23], they are tedious to perform, require a lot of training of the researchers and involve sacrifice (stomach analysis) and fistulation (oesophageal extrusa) of the animals, unless faecal samples are used. Moreover, in the case of using faecal samples, possible differential digestion of the different plant species and the large proportion of unidentifiable fragments reduce the accuracy of diet composition estimates.

Another methodology that has been used for studying diet selection of herbivores is the near-infrared reflectance spectroscopy of faeces (F.NIRS) [2427]. This methodology involves the association between faecal spectra with that of diets consumed, i.e. measurements of the reflectance of light between 700 and 2500 nm (for more details, see Dixon and Coates [28]). This spectrum gives a specific signature depending on the presence, character and number of important chemical bonds, such as OH, NH and CH [28]. According to Swain and Friend [29], one of the major limitations pointed out to NIRS applications (i.e. estimation of feed intake, digestibility and diet composition) is the need to have accurate calibration equations based on known and estimated nutritional parameters that will obviously vary for each specific situation (vegetation community). Nevertheless, these authors recognized the usefulness of this technique in identifying the presence of a specific feed item.

Results obtained by Ferreira et al. [18] suggest large variation in the spatial choice (i.e. plant communities where to graze) between animal species within a day and throughout the grazing season. The nutritive value, availability and the spatial distribution of the feed resources, and the distance to water and slope are major factors influencing grazing distribution patterns [30]. Early studies used visual field observations to assess these temporal and spatial modifications of rangeland use by both domestic and wild herbivores [30]. The utilization of recent available telemetry techniques can help grazing scientists to assess landscape vegetation preferences of herbivores [29], increasing the number of observed animals and reducing significantly the labour and allowing the collection of high-quality and unbiased data over a 24-h period. Identification of the preferred grazing sites can be accomplished by using telemetry devices, as global positioning systems (GPS). These devices are able to fine-scale spatio-temporal location data [31] with a spatial accuracy of <5 m [32] depending on the telemetry devices. This information together with data on the spatial arrangement of the plant communities can be used to assess the animals’ patch selection. According to Swain and Friend [29], the spatial arrangement of vegetation (number and size of the patches) will determine the level of local accuracy needed, i.e. small patches in larger number will need a higher accuracy of location data. In a recent study, Thompson et al. [33] used GPS collars to spatially register cattle location, and based on this, data were able to assess their activities (grazing, travelling or resting) on distinct plant communities of a rangeland, using an algorithm developed to classify cattle activity. Hebblewhite and Haydon [31] referred that the high cost of GPS collars that depends on its features (i.e. battery size, longevity, programmability, remote data access) has led researchers to opt for using fewer GPS units, limiting statistical inference. According to these authors, collar failures that could range from 5 to 50% of the units reduce even further sample size. In addition to these shortcomings, this method does not allow to quantify or estimate diet composition.

Analysis of stable carbon isotopes in animal faeces has also been used to discriminate C3 and C4 plants on the diet selected by domestic [3137] and wild herbivores [38]. This methodology is based on differences between plants with different photosynthesis pathways in fractioning of 13C, with C3 plants discriminating more against the heavier isotope 13C in favour of 12C than C4 plants. This results in different 13C:12C ratios that are expressed as δ13C relative to the 13C:12C ratio of the international Vienna Pee Dee Belemnite standard. Using these markers De Smet et al. [39] were able to estimate accurately the proportion of C4 plant material in the diet analysing stable carbon isotope ratios (δ13C value) in different tissues (blood, plasma, liver, kidney fat, hair, muscle and ruminal contents) taken from beef animals at slaughter. Nevertheless, Dove and Mayes [21] pointed out some limitations to this technique: (1) limited to situations where C4 plants are present, for example, tropical grazing systems; (2) when using faecal samples, differential recovery of feeds in faeces may lead to underestimation of those of higher digestibility; and (3) possible effect of faecal endogenous carbon on the faecal carbon isotope ratio.

Alternatively, plant-wax components, especially alkanes and other wax components, such as long-chain alcohols and long-chain fatty acids, have been suggested as possible markers to estimate diet composition. The main advantages of using these markers is the fact that for their quantification the same analytical procedure is used on samples of the diet components and animal faeces, reducing labour and analytical error. Moreover, it provides the necessary information for the estimation of diet composition, digestibility and intake for each individual, therefore accommodating possible differences between individuals [21].

3. Epicuticular compounds

The aerial surfaces of most higher plants are covered by a layer of (epicuticular) wax that is a complex mixture of hydrophobic compounds such as long-chain fatty acids, aldehydes, alcohols, triterpenes, sterols, ketones, esters, flavonoids and alkanes [40]. According to Dove and Mayes [41], the chemical composition of this layer varies within plant species and plant parts, with leaves and floral parts tending to present higher wax concentrations than stems [21]. This layer has multiple functions, being the first line of protection between plants and the environment, acting as hydrophobic barriers, limiting nonstomatal water loss, and may constitute a defence mechanism against bacterial and fungal pathogens and other stress agents [40]. According to Eigenbrode and Espelie [42], it also plays an important role in the plant-insect interactions, repelling or attracting them.

Although the first studies on the possible use of epicuticular compounds as faecal markers were carried out with long-chain fatty acids (LCFAs) by Body and Hansen [43] and Grace and Body [44], alkanes are the ones most widely studied and applied in field studies due to their relative inertness and simplicity of analysis [21]. Alkanes present in the epicuticular mixture differ in carbon-chain length, varying from 21 to 37 carbon atoms [45]; those with odd number of carbon atoms represent more than 90% of the total content. Generally, the most abundant are the n-nonacosane (C29), n-untriacontane (C31) and n-tritriacontane (C33) [22, 45]. The alkanes with less than 25 and more than 35 carbon atoms are present in very low concentrations. The alkane content varies between plant species ( Table 1 ), plant parts and even cultivars of the same species [46, 47], plant stages of maturity and climatic conditions. In general, most of the herbaceous species, especially tropical forage species [48, 49], but also some shrub species (e.g. U. gallii, [50]), are known to possess very low alkane concentrations.

Speciesn-Alkanes (mg/kg DM)
C25 C26 C27 C28 C29 C30 C31 C32 C33
Lolium perenne[50]
Lolium multiflorum 33.55.456.68.6150.813.1181.65.123.7[51]
Lolium rigidum 17.27.651.017.7254.022.9411.07.6[52]
Festuca arundinacea[53]
Holcus lanatus 133.615.8111.521.8225.323.8178.012.047.2[51]
Phalaris aquatica[52]
Nardus stricta[54]
Leymus chinensis[55]
Leymus dasystachys[55]
Elymus sibiricum[55]
Trifolium repens 16.43.838.211.3170.016.6206.97.322.2[50]
Trifolium striatum[53]
Trifolium arvensis 30.29.2122.733.9915.240.7314.220.932.5[51]
Trifolium subterraneum 4.94.752.318.1361.010.980.86.0[52]
Vicia sativa 15.83.767.012.3204.216.3502.815.129.7[51]
Ornithopus compressus 17.45.340.39.7570.18.460.81.110.7[51]
Ulex gallii[50]
Calluna vulgaris 15.89.975.426.6289.935.1939.858.8685.5[54]
Erica cinerea[54]
Erica umbellata 17.67.751.212.9239.730.9580.635.9235.7[54]
Erica arborea 14.54.667.821.4408.793.71625.8133.9662.2[50]
Erica tetralix[56]
Vaccinium myrtillus[56]

Table 1.

Alkane concentrations (mg/kg DM) of several herbaceous and shrub plant species.

As can be observed in Table 1 , differences in the alkane profiles between plant species occur in terms of absolute concentrations and relative proportions of the individual alkanes in the total content. Dove et al. [47] studied the effect of the plant species, age and part of the plant on the alkane profiles of different pasture species (Phalaris aquatica, L. perenne, T. repens, Trifolium subterraneum subsp. subterraneum, T. subterraneum subsp. yanninicum and Medicago sativa) and observed that species explained 85% and date of harvest only 6% of total variation. Differences in the alkane content between plant parts in the same pasture species were observed by Dove et al. [47]. Higher concentrations were found in the leaf than in the stem fraction. Also, much higher concentrations presented by the inflorescence of the perennial ryegrass (L. perenne) and by the flower of the white clover (T. repens) should be pointed out [47]. Less evident is the effect of age/stage of development in the alkane content of plant species. As stated above, the influence of the harvest date on the alkane content of the plant species studied by Dove et al. [47] accounted for only 5.7% of total variance. The results obtained by Oliveira et al. [57] indicate a decrease with age in the concentrations of C33 and C35 of hays of Pennisetum purpureum (C33 r = −0.97; C35 r = −0.99). Similar results were obtained by Laredo et al. [48] in leaves of Pennisetum glaucum (C33 r = −0.81; C35 r = −0.85) and Sorghum sp. (C33 r = −0.96; C35 r = −0.95) as age increased. However, opposite results were obtained by Smith et al. [58] when evaluating the effect of season of harvest on alkane concentrations of 40 common rangeland grasses found in Southern Africa. These authors did not observe a significant change in alkane concentrations either in leaf or stem components of the plant species between dry and wet seasons, suggesting that differences in alkane concentrations in whole plant samples could result from differences in the proportions of plant parts that present different alkane patterns.

Other epicuticular compounds, namely long-chain fatty alcohols (LCFAs) [5963] and LCOH [53, 6466], have also been suggested as possible diet composition markers. Also, alkenes (unsaturated aliphatic hydrocarbons) were tested with success by Dove and Oliván [67] to estimate diet composition of sheep fed with different proportions of chaffed perennial ryegrass and unpelleted sunflower meal labelled with beeswax. These epicuticular compounds have the advantage over any other possible markers as the separation and quantification of these wax components can be an extension of the alkane procedure, not adding much more analytical work [59].

It should be noted that, as stated by Dove and Mayes [21], all studies have been based on total LCFA and LCOH concentrations (i.e. free plus esterified LCFA and LCOH), as a result of the cleavage of wax esters promoted by the saponification of samples with ethanolic KOH (1 M) in the extraction process. The LCFAs present in the epicuticular waxes are mainly mixtures of straight-chain saturated compounds [41] with an even number of carbons ( Table 2 ). Within the LCFA that can be detected in animal faeces, those with carbon-chain lengths between C22 and C34 are suitable for diet composition estimation as they are exclusively associated with plant epicuticular waxes and present high recovery in animal faeces [41, 60]. Various studies have shown clear differences in the LCFA profiles between different plant species [41, 6063, 68, 69], making them useful as diet composition markers. In general, individual and total LCFA concentrations of plant species are much higher than those found for the alkanes, especially for the herbaceous species [60, 62]. In fact, Ferreira et al. [60] and Lin et al. [69] observed that the majority of LCFAs with even-chain length in herbaceous species have concentrations above 100 mg/kg DM, whereas only a few alkanes exceeded this value. Also, Ali et al. [68] and Lin et al. [69] found total LCFA concentrations that were in average 10 times greater than the total alkane concentrations of 25 different rangeland species from Sudan and native Chinese grass species (Leymus chinensis, Leymus dasystachys and Elymus sibiricum), respectively. As also found for alkanes, differences between plant parts can also be observed in their LCFA profiles. Although there is limited information on possible differences between plant parts in their LCFA profiles, results obtained by Ferreira et al. [60] indicated a trend for the leaf/stem fraction of L. perenne to present higher concentrations on the longer (>25 carbon atoms) LCFA than the inflorescence fraction.

SpeciesEven-chain fatty acids (mg/kg DM)
Lolium perenne 514.3381.9559.2396.3287.1128.232.2[60]
Leymus chinensis 212.0247.0322.091.0159.0152.0144.0[69]
Leymus dasystachys 207.0346.0229.0171.0338.0191.055.0[69]
Elymus sibiricum 196.0257.0217.0169.0165.0114.055.0[69]
Trifolium repens 612.9715.8607.9792.4440.464.71.2[60]
Ulex gallii 447.2308.9128.0114.1133.925.50.2[60]
Agrostis-Poa 1 324.2249.5522.6195.9156.797.642.3[63]
Poa spp. 2 236.1156.7366.6120.145.324.06.5[63]
Heather 3 549.9485.6482.7550.0432.8394.3105.7[63]
Calluna vulgaris 148.0347.0255.0195.0199.0168.06.031.0[68]
Erica arborea 645.2292.7215.0434.1782.9528.7138.5[60]
Vaccinium myrtillus 211.0179.0140.0128.0325.01132.0176.06.0[68]

Table 2.

Even-chain fatty acid concentrations (mg/kg DM) of several herbaceous and shrub plant species.

1Leaf fractions.

2Flowerstem fractions.

3Composed of Erica umbellata (0.76), Erica cinerea (0.16) and Calluna vulgaris (0.08).

Similarly to the LCFA, free LCOHs found in epicuticular wax of plant species are straight-chain saturated compounds with an even number of carbons within the same range of carbon-chain length referred to the LCFA (C20–C34) ( Table 3 ). They are mainly primary alcohols, although many conifers present high concentrations of the odd-chain secondary alcohol 10-nonacosanol (C29) [41]. As observed for the other epicuticular markers, LCOH profiles vary among plant species [52, 64, 65, 68, 69]. Generally, grass species are characterized by very high concentrations in C26 and C28 alcohols [52, 53, 64, 68], whilst C30 alcohol can be detected in large amounts in legumes [52, 64]. In general, total LCOH concentrations are within those of alkanes and LCFA, although Lin et al. [55, 69] reported a predominance of LCOH over the LCFA in L. chinensis, L. dasystachys, E. sibiricum, Stipa baicalensis, Stipa grandis and Cleistogenes squarrosa. As also found for the alkanes and LCFA, results suggest clear differences between vegetative and reproductive parts of herbaceous species. In fact, Ferreira et al. [64] indicated that the reproductive parts of L. perenne are characterized by having higher proportions of shorter LCOH than the vegetative tissues.

SpeciesEven-chain alcohols (mg/kg DM)
Lolium perenne 47.361.5162.52159.6517.4149.054.8[64]
Lolium rigidum 12.360.01751.0363.5176.1[52]
Festuca arundinacea 26.8638.8100.558.0[53]
Phalaris aquatica 14.637.82813.0134.965.3[52]
Leymus chinensis 021.073.0361.0846.0252.0116.0[69]
Leymus dasystachys 9.028.0142.03815.04418.0442.070.0[69]
Elymus sibiricum 11.014.0126.02374.0185.050.00[69]
Trifolium repens 26.535.645.7415.3167.71077.584.9[64]
Trifolium striatum 37.0214.4443.51259.3[58]
Trifolium subterraneum 23.7240.5503.9369.92141.0[53]
Ulex gallii 30.881.7189.1120.1133.4111.7153.9[64]
Calluna vulgaris 221.01190.0474.0203.0450.0829.0[56]
Erica arborea 157.9315.0210.8120.8206.0262.146.1[64]
Erica tetralix 62.0560.01124.01015.01496.04465.0[56]
Vaccinium myrtillus 271.0511.0362.0334.0383.0931.0[56]

Table 3.

Even-chain alcohol concentrations (mg/kg DM) of several herbaceous and shrub plant species.

4. Application of epicuticular compounds as biomarkers

The differences in the profiles of the epicuticular compounds mentioned above can be explored to estimate the proportions of different plant species and plant parts in different samples, such as herbage mixtures [70]; extrusa from oesophageal-fistulated animals [71] or faeces of sheep [52, 61, 65, 69, 7275], goats [50, 60, 64, 76], cattle [62, 63, 66, 76, 77] and horses [62, 66, 77]. The principle of the application of the technique is simple and relies on the comparison of marker concentrations in a mixture (extrusa, digesta or faeces) and in diet components, plant species and/or plant parts that contribute (or could contribute) to that mixture. The comparison of the marker profiles can be made using different calculation procedures. It should be pointed out that more important than choosing the calculation procedure used, it is necessary to ensure that the information used (marker profiles of the possible diet components and the resultant mixture—faeces) is as accurate as possible.

Dove [70] proposed the utilization of simultaneous equations to estimate the proportions of the possible diet components when using alkanes as diet composition markers. In order to obtain unique solutions, the number of markers used is equal to the number of diet components and to the number of equations created [22]. The result of the equations indicates the amounts of the different diet components necessary to produce 1 kg of faeces, making possible to estimate the digestibility of the estimated diet. According to Dove and Mayes [78], this calculation procedure can be used in simple dietary mixtures, being more difficult to compute in complex mixtures. The main limitation of this procedure is in situations where there are more markers than the possible diet components, being necessary to select the markers to be used in the calculations. This selection involves arbitrary choices of the markers and the loss of information provided by the markers which were not used in the calculations. Moreover, this procedure may occasionally produce meaningless biological results as negative proportions of the diet components considered in the calculations.

In order to surpass these limitations, least-squares optimization methods can be applied, for which several algorithms have been developed [71, 7981]. These calculation methodologies allow us to accommodate concentrations of different marker types (alkanes, LCFA, LCOH). The solution achieved by these algorithms attempts to minimize the squared deviations between the observed (O) marker concentrations in faeces and the concentration profile (E) arising from the diet composition estimate [21]:




where x, y and z are the proportions of components A, B and C in the diet; Fi, Ai, Bi and Ci are the concentrations of alkane i in faeces and diet components A, B and C; Fj, Aj, Bj and Cj are the concentrations of LCOH j in faeces and diet components A, B and C; Fk, Ak, Bk and Ck are the concentrations of LCFA k in faeces and diet components A, B and C; Ft, At, Bt and Ct are total alkane concentrations in faeces and diet components A, B and C; Fu, Au, Bu and Cu are total LCOH concentrations in faeces and diet components A, B and C; Fv, Av, Bv and Cv are total LCFA concentrations in faeces and diet components A, B and C. It is possible to express the individual marker concentrations in the feeds and faeces as absolute concentrations (Eq. (1)) or as proportions of the total concentration (Eq. (2)). The advantage of using concentrations instead of proportions is that x, y and z estimates using Eq. (1) are the amounts which will result in 1 kg of faeces. Thus, this information can be used to obtain an estimate of diet digestibility as


5. Major constraints to the application of biomarkers in herbivory studies

As stressed by Dove and Mayes [21], it is important to ensure that the information used in both sides of Eqs. (1) and (2) (marker patterns of diet components and animal faeces) is as accurate as possible. An important source of error, often forgotten by researchers when applying the epicuticular markers to estimate diet selection in grazing studies, is the representativeness of the hand-collected samples of the vegetation components, in terms of marker profiles. This task can be difficult to accomplish as there can be significant variations in the marker profiles between plant species and plant parts within a specific plant species, as mentioned earlier. Other aspects requiring special attention are the continuous modification of each vegetation component available in the pasture, the relationship between plant parts and its stage of maturity and, consequently, their marker patterns. For this reason, it is recommended to collect samples of the plant species corresponding to each measuring period. Another important constraint associated with feeds/plant species is their very low marker concentrations. For example, herbaceous species (L. perenne, T. repens, Pseudarrhenatherum longifolium, Agrostis capillaris, [82] and P. aquatica [83]) and some shrub species (U. gallii [82]) are characterized by having low alkane concentrations and, for that reason, are more prone to analytical errors. Ferreira et al. [60] suggested that in these situations other marker types (e.g. LCFA or LCOH) should be used.

An additional concern is the collection of representative samples of faeces in terms of marker profiles. As occurs with other types of markers, the variation within and between days in the faecal concentrations can limit the utilization of this technique. In general, this variation is observed for dosed even-chain alkanes that are used for intake estimation [49, 8491] due to their tendency to be associated with the liquid phase of the digesta. For that reason, an adaptation period of 5 days for the synthetic alkanes to reach a steady-state excretion pattern in animal faeces is generally suggested [21]. Regarding the natural markers, in grazing studies it is likely the existence of variation in the diet selected by the animals and, consequently, in feed intake, digestibility and faecal output from day to day [78]. For this reason, these authors suggest a sampling period of 5–7 days to obtain a more representative sample of faeces.

An assumption inherent to the application of the epicuticular compounds as diet composition markers is that they are totally recovered in the faeces. The results obtained in metabolic crate studies clearly indicate an incomplete recovery of alkanes [21, 73, 76], LCFA [5963] and LCOH [52, 59, 6466, 69] in the faeces of ruminant species ( Figure 3 ), suggesting a close association between the length of the carbon chain of markers and its faecal recovery. Generally, results suggest a higher faecal recovery in the LCOH than in alkanes and LCFA [59, 64]. This is possibly related to the different location of these compounds in the wax layer (i.e. alkanes in greater concentrations in the epicuticular layer, whilst primary alcohols are found in greater concentrations in the intracuticular wax [92] that could interfere with the efficiency of the extraction of these compounds and/or their absorption in the ruminants’ digestive tract [65, 69, 93].


Figure 3.

Effect of carbon-chain length on the faecal recovery (%) of alkanes [94], long-chain fatty acids (LFCA) and long-chain alcohols (LCOH) observed in ruminant species.

In several studies, the relationship between carbon-chain length and faecal recovery is better described by curvilinear functions for alkanes [60, 83, 93], LCFA [60, 61] and LCOH [65] as a result of the decrease in the difference of faecal recovery of markers with adjacent carbon-chain length with increasing carbon-chain length. By contrast, other studies have reported a linear association between carbon-chain length and faecal recovery in alkanes [75, 76, 96] and LCOH [64]. It seems that the association is dependent on the feeds/plant species comprising the diets and has an important effect on the accuracy of diet composition estimates on ruminant species. In fact, if uncorrected marker faecal concentrations are used in the calculations (Eq. (1) or (2)), estimates of diet composition will be biased towards those feeds/plant species with a predominance of longer carbon-chain length markers that have higher faecal recovery rates. For that reason, a suitable correction of marker faecal concentrations for incomplete faecal recovery, before applying them for diet composition estimation in grazing animals is generally suggested. Nevertheless, in situations where feeds/plant species do not have any chain-length bias, the effect of the recovery correction has little effect on the accuracy of diet composition estimates [60].

Data on marker faecal recoveries can be obtained in metabolic cage studies with animals fed on different mixtures of feeds/plant species that are available for each specific situation. It should be noted that in complex situations in terms of number of possible diet components, it will be difficult to decide which combination of plant species and/or plant parts will reflect the diet selected by each different animal species. For alkanes, Dove and Mayes [21] suggested another option that consists of dosing a range of synthetic even-chain alkanes and collecting the total faecal production by wearing faeces bags and calculating the faecal recoveries of natural odd-chain alkanes by interpolation. Nevertheless, it has been found that the synthetic dosed alkanes may have higher recoveries than those expected from interpolation of adjacent natural odd-chain alkanes [82, 87, 98].

For non-ruminant species such as horses [97, 99102], pigs [103, 104], mountain hares [105] and pigeons [106], marker faecal recoveries seem to be unrelated to their carbon-chain length ( Figure 4 ), indicating that these markers behave differently in the digestive tract of ruminants and non-ruminants, especially those with lower carbon-chain length. In fact, the comparison of faecal recovery data between ruminant and non-ruminant species for alkanes [77], LCFA [62] and LCOH [66] indicates a greater disappearance of the shorter markers in the gut of ruminants than in non-ruminants. The site and the mechanisms underlying marker losses in the animal gastrointestinal tract are still far from being completely elucidated. Earlier studies undertaken by Mayes et al. [107] suggested that the disappearance of the dosed alkanes C28, C32 and C36 occurred mainly in the small intestine in sheep. More recently, Keli et al. [25] also suggested that alkane disappearance should mainly occur in the small intestine as they were not be able to find evidences of rumen microorganisms’ capability to synthesize or metabolize alkanes in in vitro conditions. By contrast, Ohajuruka and Palmquist [108] found that the loss of dosed C32 alkane in dairy cows occurs mainly in the rumen.


Figure 4.

Effect of carbon-chain length on the faecal recovery (%) of alkanes [109111], long-chain fatty acids (LFCA) and long-chain alcohols (LCOH) observed in non-ruminant species.

The lack of a clear relationship between the carbon-chain length of the epicuticular compounds and their faecal recovery in non-ruminant species has an important effect on diet composition estimates. In fact, Ferreira et al. [77] were not be able to observe an increase in the accuracy of diet composition estimates in horses when alkane faecal concentrations corrected for their incomplete recovery were used. This lower dependence of markers for a suitable faecal recovery correction was also found in LCOH and LCFA by López López et al. [66] and Ferreira et al. [62], respectively, although in their cases a linear association between carbon-chain length and faecal recovery was observed. These results indicate that, for this animal species, accurate estimates of diet composition can be obtained even when raw data of the faecal concentrations of these epicuticular compounds (i.e. without previous corrections of the faecal concentrations) are used.

Another important constraint that limits a wider applicability of epicuticular compounds as markers in grazing studies is that their faecal recovery may depend on the diet composition, compelling researchers to calculate faecal recoveries for each specific situation (i.e. diet composition), making it impossible to use recovery data available in literature. This effect was observed in several studies performed with alkanes [50, 83], LCOH [64, 65, 69] and LCFA [60, 61, 69], whilst others were not be able to detect it [52, 73, 76, 96]. According to some authors [64, 69, 96], this inconsistency may be due to the particular plant species comprising the diets. Lin et al. [69, 75], using sheep fed distinct grass species (E. sibiricum, L. chinensis or L. dasystachys) obtained different faecal recoveries of LCOH, LCFA and alkanes. According to the same authors, these results could be explained by differences among plant species in their plant cuticular wax morphology, influencing the level of extraction and the absorption of these compounds in the digestive tract of animals. Diet digestibility may also explain these differences in the faecal recovery of the epicuticular compounds among different diets. In fact, a general tendency for higher faecal recoveries of alkanes [50, 74, 112] and LCOH [64] in diets with lower digestibility was observed. Lower accessibility to the cuticular waxes of those feeds as a result of a higher association of cuticle with cell wall components [113] may explain the lower availability of epicuticular compounds to be absorbed in the digestive tract of animals.

The first epicuticular compounds suggested as diet composition markers were the alkanes [45], and the limited number of components (e.g. plant species and/or plant parts) that can be discriminated in the diet that is restricted to the number of n-alkanes available was soon recognized. The number of alkanes available for diet composition calculations is generally limited to 9 (C25–C33), due to the higher potential analytical error associated with those of very low concentrations both in plants and in faeces, which may contribute to the discrepancy in sum of squares in the calculation method [78, 95]. Moreover, it is accepted that the increase of the number of diet components to be discriminated will likely result in less accurate diet composition estimates, as it increases the likelihood that an observed alkane pattern in faeces may result from different combinations of diet components [21]. To overcome these limitations, a possible approach to obtain reliable diet composition estimates is to increase the number of ‘discriminators’ by combining the use of alkanes with other plant-wax markers, such as alkenes [67], LCOH [52, 53, 64, 69] and LCFA [5963]. According to Bugalho et al. [53], combination of markers should only be performed when additional discriminatory information is provided. Combination of different marker types may improve the accuracy of diet composition estimates as markers with greater concentrations, less prone to analytical error, can be selected in situations where the possible dietary feed items have similar alkane profiles. Moreover, it is likely that the use of a greater number of markers provides a more specific ‘fingerprint’ for a particular plant component [60]. This is also very important even when the number of plant species to be discriminated is low, but they present high similarities in alkane profiles, making it difficult to discriminate them.

In several studies, it was possible to observe an increase in the accuracy of diet composition estimates when combining two [52, 60, 114] or three marker types [6466, 68, 69]. It should be pointed out that the combination of epicuticular compounds does not necessarily result in more accurate estimates of diet composition [21, 53]. For example, Vargas-Jurado et al. [115] did not observe an improvement of the predictions of the composition of mixtures of tall fescue (Festuca arundinacea) and red clover (Trifolium pratense) when LCOH was combined with alkanes. Nevertheless, results obtained by Ferreira et al. [60, 64, 65] suggest that the combination of markers reduce their dependence on accurate faecal-correction data for an accurate diet composition estimation as more specific fingerprints of each plant species are achieved, increasing their ability to discriminate them.

Another approach that is suggested when the number of possible diet components is high is to decrease the number of possible diet components by pooling the available plant species into groups [81, 116]. These groups are formed by plant species with similar marker profiles, based on multivariate statistical analysis, that are then treated as dietary components in the calculations. One aspect that needs particular attention is the fact that the accuracy of diet composition estimates can be influenced by different availability or selectivity levels of some plant species within each group, especially if the marker profile of a particular plant species is distinct from the mean marker profile of the group in which that species is included [51]. As pointed out by Ferreira et al. [116], feeding selectivity effect will depend on the particular species that could be selected within the group and on the similarity in the marker profile of the plant species of the dietary group. Bugalho et al. [51] did not found any feeding selectivity effect within a group of 19 herbaceous species on diet composition estimates of red deer. Similar results were observed by Ferreira et al. [116] when applying different levels of feeding selectivity to a dietary group formed by heather species (C. vulgaris, Erica cinerea, Erica umbellata and Erica australis) with similar alkane profile. By contrast, when the same procedure was applied to a dietary group formed by three grass species (L. perenne, P. longifolium and A. capillaris), significant modifications of the proportions of each dietary group were observed. A decrease in feeding selectivity effect can be achieved by the formation of more uniform dietary groups in terms of marker profiles. However, Ferreira et al. [63] observed higher levels of accuracy of diet composition estimates when considering all plant species that animals had at their disposal compared to its grouping according to the similarity of alkane and LCFA profiles.

The exclusion of plant species based on preliminary information is another approach suggested by Dove and Mayes [21] to reduce the number of possible diet components. The observation of the animals’ feeding behaviour, plant-derived data or information based on other methodologies that indicate the rejection of a particular plant species and/or vegetation community, can help the researcher to use more accurate data on the plant species that should be considered in the calculations.

The utilization/combination of different types of markers has also advantages in less complex plant communities (i.e. lower number of plant species to be discriminated), by giving the opportunity to the researcher to choose those with higher concentrations less prone to measurement errors in their analytical determination [21]. According to Charmley and Dove [83] and Ferreira et al. [60, 61], the utilization of markers with low concentrations can turn discrimination of plant species more difficult and may result in less accurate estimates of diet composition. In fact, Oliván et al. [82] attributed the difficulties in distinguishing three grass species (L. perenne, P. longifolium and A. capillaris) and gorse to their low alkane concentrations. Also, Charmley and Dove [83] had difficulties in obtaining accurate estimates of diet composition when P. aquatica (plant species characterized by very low alkane concentrations) was a component of diets fed to sheep. In our opinion, the exclusion of markers based on their low concentrations should be performed with caution as, in some situations, they may discriminate better plant species than those with higher concentrations. Thus, Dove and Mayes [21] suggested that the balance between the capability of markers to discriminate plant species and the level of potential analytical error should be considered when choosing markers.

6. Conclusions

Taking into account all the data presented in this chapter, it is certain that the application of the epicuticular compounds as faecal markers can improve our knowledge on the grazing behaviour, particularly diet selection, of free-ranging herbivore species under different vegetation conditions. Although some shortcomings can be pointed out to these faecal markers, namely the variation of profiles within plant species and morphological parts, lack of inertness in the digestive tract of ruminant species and, for that reason, the need for a suitable recovery correction of their faecal concentrations, they have been used quite successfully. Its application allows to overcome major limitations recognized to the traditional techniques in terms of accuracy and extent of the results (i.e. identification of plant species and/or plant parts), animal welfare issues (i.e. avoid the need for fistulated animals; lower disturbance of animals compromising its normal grazing behaviour) and intensive labour. The combination of different maker types (alkanes, LCOH, LCFA) seems promising to overcome the enumerated constraints and to extend their application to more complex vegetation communities. Therefore, research on the identification of other chemical compounds should continue to be developed. Finally, data obtained from different available techniques (microhistological procedures, NIRS, Fourier-transform infrared (FTIR) spectroscopy and fluorescence spectroscopy, telemetry solutions) should be integrated in order to enhance the accuracy of diet composition being selected by herbivore species. This will further improve the precision of information (i.e. possible diet components) used when applying the epicuticular markers.


This work was financed by the Portuguese Science and Technology Foundation (FCT) under the Project UID/CVT/00772/2013.


1 - Gordon IJ, Hester AJ, Festa-Bianchet M. The management of wild large herbivores to meet economic, conservation and environmental objectives. J. Appl. Ecol. 2004;41:1021–1031. DOI:10.1111/j.0021-8901.2004.00985.x
2 - Hodgson J. Grazing behaviour and herbage intake. In: Frame J, editor. Grazing. BGS Occ. Symp. No. 19;1986. pp. 51–64.
3 - Hutchings NJ, Gordon IJ. A dynamic model of herbivore-plant interactions on grasslands. Ecol. Model. 2001;136:209–222. DOI:10.1016/S0304-3800(00)00426-9
4 - Hester AJ, Gordon IJ, Baillie GJ, Tappin E. Foraging behaviour of sheep and red deer within natural heather/grass mosaics. J. Appl. Ecol. 1999;36:133–146. DOI:10.1046/j.1365-2664.1999.00387.x
5 - Hülber K, Ertl S, Gottfried M, Reiter K, Grabherr G. Gourmets or gourmands? Diet selection by large ungulates in high-alpine plant communities and possible impacts on plant propagation. Basic Appl. Ecol. 2005;6:1–10. DOI:10.1016/j.baae.2004.09.010
6 - Rook AJ, Dumont B, Isselstein J, Osoro K, WallisDeVries MF, Parente G, Mills J. Matching type of grazing animal to desired biodiversity outcomes—a review. Biol. Conserv. 2004;119:137–150. DOI:10.1016/j.biocon.2003.11.010
7 - Rook AJ, Tallowin JRB. Grazing and pasture management for biodiversity benefit. Anim. Res. 2003;52:181–189. DOI:10.1051/animres:2003014
8 - Hodgson J, Forbes TDA, Armstrong RH, Beattie MM, Hunter EA. Comparative studies of the ingestive behaviour and herbage intake of sheep and cattle grazing indigenous hill plant communities. J. Appl. Ecol. 1991;28:205–227. DOI:10.2307/2404126
9 - Celaya R, Oliván M, Ferreira LMM, Martínez A, García U, Osoro K. Comparison of grazing behaviour, dietary overlap and performance in non-lactating domestic ruminants grazing on marginal heathland areas. Livest. Sci. 2007;106:271–281. DOI:10.1016/j.livsci.2006.08.013
10 - Fraser MD, Theobald VJ, Griffiths JB, Morris SM, Moorby JM. Comparative diet selection by cattle and sheep grazing two contrasting heathland communities. Agric. Ecosyst. Environ. 2009;129:182–192. DOI:10.1016/j.agee.2008.08.013
11 - Osoro K, Oliván M, Celaya R, Martínez A. Effects of genotype on the performance and intake characteristics of sheep grazing contrasting hill vegetation communities. Anim. Sci. 1999;69:419–426.
12 - Osoro K, García U, Jáuregui BM, Ferreira LMM, Rook AJ, Celaya R. Diet selection and live-weight changes of two breeds of goats grazing on heathlands. Animal 2007;1:449–457. DOI:10.1017/S1751731107683797
13 - Hofmann RR. Evolutionary steps of ecophysiological adaptation and diversification of ruminants: comparative view of their digestive system. Oecologia 1989;78:443–457. DOI: 10.1007/BF00378733
14 - Mysterud A. The relative roles of body size and feeding type on activity time of temperate ruminants. Oecologia 1998;113:442–446. DOI:10.1007/s004420050396
15 - Pérez-Barbería FJ, Gordon IJ, Illius AW. Phylogenetic analysis of stomach adaptation in digestive strategies in African ruminants. Oecologia 2001;129:498–508. DOI:10.1007/s004420100768
16 - Van Soest PJ. Nutritional Ecology of the Ruminant. 2nd ed. Ithaca, NY: Cornell University Press; 1994. 476p.
17 - Udén P, Van Soest PJ. Comparative digestion of timothy (Phleum pratense) fibre by ruminants, equines and rabbits. Br. J. Nutr. 1982;47:267–272. DOI:10.1079/BJN19820035
18 - Ferreira LMM, Celaya R, Benavides R, Jáuregui BM, García U, Santos AS, Rosa García R, Rodrigues MAM, Osoro K. Foraging behaviour of domestic herbivore species grazing on heathlands associated with improved pasture areas. Livest. Sci. 2013;155:373–383. DOI:10.1016/j.livsci.2013.05.007
19 - Osoro K, Ferreira LMM, García U, Martínez A, Celaya R. Forage intake, digestibility and performance of cattle, horses, sheep and goats grazing together on an improved heathland Anim. Prod. Sci. DOI:10.1071/AN15153
20 - Holechek JL, Vavra M, Pieper RD. Botanical composition determination of range herbivore diets: a review. J. Range Manage. 1982;35:309–315. DOI:10.2307/3898308
21 - Dove H, Mayes RW. Using n-alkanes and other plant wax components to estimate intake, digestibility and diet composition of grazing/browsing sheep and goats. Small Rumin. Res. 2005;59:123–139. DOI:10.1016/j.smallrumres.2005.05.016
22 - Dove H, Mayes RW. Plant wax components: A new approach to estimating intake and diet composition in herbivores. J. Nutr. 1996;126:13–26.
23 - Mayes RW, Dove H. Measurement of dietary nutrient intake in free-ranging mammalian herbivores. Nutr. Res. Rev. 2000;13:107–138. DOI:10.1079/095442200108729025
24 - Walker JW, Campbell ES, Lupton CJ, Taylor CA Jr, Waldron DF, Landau S. Effects of breed, sex, and age on the variation and ability of Fecal near-infrared reflectance spectra to predict the composition of goat diets. J. Anim. Sci. 2007;85:518–526. DOI:10.2527/jas.2006-202
25 - Keli A, Mayes RW, de Vega A. In vitro studies of the metabolism of [14C]-n-alkanes using ruminal fluid of sheep as substrate. Animal 2008;2:1748–1752. DOI:10.1017/S1751731108003108
26 - Decruyenaere V, Buldgen A, Stilmant D. Factors affecting intake by grazing ruminants and related quantification methods: a review. Biotechnol. Agron. Soc. Environ. 2009;13:559–573.
27 - Landau SY, Dvash L, Roudman M, Muklada H, Barkai D, Yehuda Y, Ungar ED. Faecal near-IR spectroscopy to determine the nutritional value of diets consumed by beef cattle in east Mediterranean rangelands. Animal 2016;10:192–202. DOI:10.1017/S175173111500169X
28 - Dixon RM, Coates DB. Review: Near infrared spectroscopy of faeces to evaluate the nutrition and physiology of herbivores. J. Near Infrared Spectrosc. 2009;17:1–31. DOI:10.1255/jnirs.822
29 - Swain DL, Friend MA. Opportunities for telemetry techniques in studies on the nutritional ecology of free-ranging domesticated ruminants. Animal 2013;7(s1):123–131. DOI:10.1017/S1751731112000870
30 - Vallentine JF. Grazing Management. 2nd ed. San Diego, CA: Academic Press; 2001. 659 p.
31 - Hebblewhite M, Haydon DT. Distinguishing technology from biology: a critical review of the use of GPS telemetry data in ecology. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2010;365:2303–2312. DOI: 10.1098/rstb.2010.0087
32 - Swain DL, Wark T, Bishop-Hurley GJ. Using high fix rate GPS data to determine the relationships between fix rate, prediction errors and patch selection. Ecol. Model. 2008;212:273–279. DOI: 10.1016/j.ecolmodel.2007.10.027
33 - Thompson DJ, Wheatley BJ, Church JS, Newman R, Walker J. Comparing grazing and resting electivity of beef cattle for BC bunchgrass communities using GPS collars. Can. J. Anim. Sci. 2015;95:499–507. DOI:10.4141/cjas-2014-116
34 - Jones RJ, Ludlow MM, Troughton JH, Blunt CG. Estimation of the proportion of C3 and C4 plant species in the diet of animals from the ratio of natural 12C and 13C isotopes in the faeces. J. Agric. Sci. 1979;92:91–100. DOI: 10.1017/S0021859600060536
35 - García SC, Holmes CW, Hodgson J, MacDonald A. The combination of the n-alkanes and 13C techniques to estimate individual dry matter intakes of herbage and maize silage by grazing dairy cows. J. Agric. Sci. 2000;135:47–55.
36 - Sponheimer M, Robinson T, Ayliffe L, Passey B, Roeder B, Shipley L, Lopez E, Cerling T, Dearing D, Ehleringer J. An experimental study of carbon-isotope fractionation between diet, hair, and feces of mammalian herbivores. Can. J. Zool. 2003;81:871–876. DOI: 10.1139/Z03-066
37 - Norman HC, Wilmot MG, Thomas DT, Masters DG, Revell DK. Stable carbon isotopes accurately predict diet selection by sheep fed mixtures of C3 annual pastures and saltbush or C4 perennial grasses. Livest. Sci. 2009;121:162–172. DOI: 10.1016/j.livsci.2008.06.005
38 - Botha S, Stock W. Stable isotope composition of faeces as an indicator of seasonal diet selection in wild herbivores in southern Africa. S. Afr. J. Sci. 2005;101:371–374
39 - De Smet S, Balcaen A, Claeys E, Boeckx P, van Cleemput O. Stable carbon isotope analysis of different tissues of beef animals in relation to their diet. Rapid Commun. Mass Spectrom. 2004;18:1227–1232. DOI: 10.1002/rcm.1471
40 - Post-Beittenmiller D. Biochemistry and molecular biology of wax production in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996;47:405–430. DOI:10.1146/annurev.arplant.47.1.405
41 - Dove H, Mayes RW. Protocol for the analysis of n-alkanes and other plant-wax compounds and for their use as markers for quantifying the nutrient supply of large mammalian herbivores. Nat. Protoc. 2006;4:1680–1697. DOI:10.1038/nprot.2006.225
42 - Eigenbrode SD, Espelie KE. Effects of plant epicuticular lipids on insect herbivores. Annu. Rev. Entomol. 1995;40:171–194. DOI:10.1146/annurev.en.40.010195.001131
43 - Body DR, Hansen RP. The occurrence of C13 to C31 branched-chain fatty acids in the faeces of sheep fed rye grass, and of C12 to C34 normal acids in both the faeces and the rye grass. J. Sci. Food Agric. 1978;29:107–14. DOI:10.1002/jsfa.2740290206
44 - Grace ND, Body DR. The possible use of long chain (C19-C32) fatty acids in herbage as an indigestible faecal marker. J. Agric. Sci. 1981;97:743–745. DOI: 10.1017/S0021859600037126
45 - Dove H, Mayes RW. The use of plant wax alkanes as markers substances in studies of the nutrition of herbivores: a review. Aust. J. Agric. Res. 1991;42:913–957. DOI:10.1071/AR9910913
46 - Baker SK, Klein L. Potential to use n-alkanes in plant cuticular waxes to discriminate plant parts of subterranean clovers eaten by ruminants. Proc. Aust. Soc. Anim. Prod. 1994;20:419. Available from: [Accessed: 2016-07-12]
47 - Dove H, Mayes RW, Freer M. Effects of species, plant part, and plant age on the n-alkane concentrations in the cuticular wax of pasture plants. Aust. J. Agric. Res. 1996;47:1333–47. DOI:10.1071/AR9961333
48 - Laredo MA, Simpson GD, Minson DJ, Orpin CG. The potential for using n-alkanes in tropical forages as a marker for determination of dry matter intake by grazing ruminants. J. Agric. Sci. 1991;117:355–361. DOI: 10.1017/S0021859600067101
49 - Morais JAS, Berchielli TT, de Veja A, Queiroz MFS, Keli A, Reis RA, Bertipaglia LMA, Souza SF. The validity of n-alkanes to estimate intake and digestibility in Nellore beef cattle fed a tropical grass (Brachiaria brizantha cv. Marandu). Livest. Sci. 2011;135:184–192. DOI: 10.1016/j.livsci.2010.07.004
50 - Ferreira LMM, Oliván M, Rodrigues MAM, García U, Osoro K. Validation of the alkane technique to estimate diet selection of goats grazing heather-gorse vegetation communities. J. Sci. Food Agric. 2005;85:1636–1646. DOI:10.1002/jsfa.2162
51 - Bugalho M, Mayes RW, Milne JA. The effects of feeding selectivity on the estimation of diet composition using the n-alkane technique. Grass Forage Sci. 2002;57:224–231. DOI: 10.1046/j.1365-2494.2002.00320.x
52 - Dove H, Charmley E. Using the alkanes and long-chain alcohols of plant cuticular wax to estimate diet composition and the intakes of mixed forages in sheep consuming a known amount of alkane-labelled supplement. Animal. 2008;2:1474–1485. DOI:10.1017/S1751731108002735
53 - Bugalho MN, Dove H, Kelman W, Wood JT, Mayes RW. Plant wax alkanes and alcohols as herbivore diet composition markers. J. Range Manage. 2004;57:259–268. DOI:10.2458/azu_jrm_v57i3_bugalho
54 - Oliván M, Osoro K. Utilization of the n-alkane technique to study intake and diet selection of grazing ruminants: review. ITEA Inf. Tec. Econ. Agrar. 1997;93A:193–208.
55 - Lin LJ, Luo HL, Wang H, Zhang YJ, Shu B. Evaluation of long-chain alcohols and fatty acids, in combination with alkanes, as markers in the estimation of the composition of four herbages in mixtures. Grass Forage Sci. 2008;64;19–25. DOI:10.1111/j.1365-2494.2008.00663.x
56 - Fraser MD, Theoblad VJ, Moorby JM. Determining diet composition on complex swards using n-alkanes and long-chain fatty alcohols. Ecol. Appl. 2006;16:1901–1910. DOI: 10.1890/1051-0761(2006)016[1901:DDCOCS]2.0.CO;2
57 - Oliveira DE, Prates ER, Peralba MCR. Identification and quantification of n-alkanes of forage plant waxes. Rev. Bras. Zootec. 1997;26:881–886.
58 - Smith DG, Mayes RW, Raats JG. Effects of species, plant part, and season of harvest on n-alkane concentrations in the cuticular wax of common rangeland grasses from southern Africa. Aust. J. Agric. Res. 2001;52:875–882. DOI:10.1071/AR00032
59 - Ali HAM, Mayes RW, Lamb CS, Hector BL, Verma AK, Orskov ER. The potential of long-chain fatty alcohols and long-chain fatty acids as diet composition markers: development of methods for quantitative analysis and faecal recoveries of these compounds in sheep fed mixed diets. J. Agric. Sci. 2004;142:71–78. DOI:10.1017/S0021859604004034
60 - Ferreira LMM, Carvalho S, Falco V, Celaya R, García U, Santos AS, Rodrigues MAM, Osoro K. Assessment of very long-chain fatty acids as complementary or alternative natural fecal markers to n-alkanes for estimating diet composition of goats feeding on mixed diets. J. Anim. Sci. 2009;87:2732–2745. DOI:10.2527/jas.2008-1718
61 - Ferreira LMM, Celaya R, Falco V, Oliván M, Santos AS, Guedes C, Rodrigues MAM, Osoro K. Evaluation of very long-chain fatty acids and n-alkane epicuticular compounds as markers for estimating diet composition of sheep fed heathland vegetation species. Anim. Feed Sci. Technol. 2010;156:75–88. DOI: 10.1016/j.anifeedsci.2010.01.007
62 - Ferreira LMM, Celaya R, Santos AS, Falco V, Guedes C, Rodrigues MAM, Osoro K. Comparison of long-chain fatty acids and alkanes as markers to estimate diet composition of equines and cattle consuming heathland vegetation species. Livest. Sci. 2010;131:260–271. DOI:10.1016/j.livsci.2010.04.011
63 - Ferreira LMM, Celaya R, Santos AS, Falco V, Guedes C, Rodrigues MAM, Osoro K. The utilization of long-chain fatty acids as markers for diet composition estimates in ruminants: effects of animal species, diet composition and marker combination. Grass Forage Sci. 2011;66:183–195. DOI:10.1111/j.1365-2494.2010.00774.x
64 - Ferreira LMM, Celaya R, Santos AS, Guedes CMV, Rodrigues MAM, Mayes RW, Osoro K. Evaluation of long-chain alcohols as diet composition markers in goats grazing heathland areas. Animal 2012;6:683–692. DOI:10.1017/S1751731111001881
65 - Ferreira LMM, Celaya R, Santos AS, Mayes RW, Rodrigues MAM, Osoro K. Application of long-chain alcohols as diet composition markers in sheep fed on grass–clover/heather–gorse plant species. Grass Forage Sci. 2015;70:30–43. DOI:10.1111/gfs.12083
66 - López López CL, Celaya R, Santos AS, Rodrigues MAM, Osoro K, Ferreira LMM. Application of long-chain alcohols as faecal markers to estimate diet composition of equines and cattle fed with herbaceous and woody species. Animal 2015;9:1786–1794. DOI: 10.1017/S1751731115001196
67 - Dove H, Oliván M. The possible use of the alkenes (unsaturated hydrocarbons) of plant cuticular wax as diet composition markers in sheep. In: Sandoval-Castro CA, DeB Hovell FD, Torres-Acosta JFJ, Ayala-Burgos A, editors. Herbivores. The Assessment of Intake, Digestibility and the Roles of Secondary Compounds. England: Nottingham University Press; 2006. pp 1–9.
68 - Ali HAM, Mayes RW, Hector BL, Ørskov ER. Assessment of n-alkanes, long-chain fatty alcohols and long-chain fatty acids as diet composition markers: the concentrations of these compounds in rangeland species from Sudan. Anim. Feed Sci. Technol. 2005;121:257–271. DOI: 10.1016/j.anifeedsci.2005.02.026
69 - Lin LJ, Luo HL, Zhang YJ, Wang H, Shu B, Hong FZ. The potential use of long-chain alcohols and fatty acids as diet composition markers: factors influencing faecal recovery rates and diet composition estimates in sheep. Animal. 2009;3:1605–1612. DOI:10.1017/S1751731109990401
70 - Dove H. Using the n-alkanes of plant cuticular wax to estimate the species composition of herbage mixtures. Aust. J. Agric. Res. 1992;43:1711–1724. DOI:10.1071/AR9921711
71 - Salt CA, Mayes RW, Colgrove PM, Lamb CS. The effects of season and diet composition on the radiocaesium intake by sheep grazing on heather moorland. J. Appl. Ecol. 1994;31:125–136. DOI:10.2307/2404605
72 - Lewis RM, Magadlela AM, Jessop NS, Emmans GC. The ability of the n-alkane technique to estimate intake and diet choice of sheep. Anim. Sci. 2003;77:319–327.
73 - Valiente OL, Delgado P, de Vega A, Guada JA. 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. DOI:10.1071/AR02221
74 - Ferreira LMM, Oliván M, Celaya R, Garcia U, Rodrigues MAM, Osoro K. The use of the alkane technique to estimate diet selection of sheep grazing grass-clover/heather-gorse vegetation communities. J. Sci. Food Agric. 2007;87:274–285. DOI:10.1002/jsfa.2717
75 - Lin LJ, Luo HL, Zhang YJ, Shu B. The effects, in sheep, of dietary plant species and animal live weight on the faecal recovery rates of alkanes and the accuracy of intake and diet composition estimates obtained using alkanes as faecal markers. J. Agric. Sci. 2007;145:87–94. DOI:10.1017/S002185960600654X
76 - Brosh A, Henkin Z, Rothman SJ, Aharoni Y, Orlov A, Arieli A. Effects of faecal n-alkane recovery in estimates of diet composition. J. Agric. Sci. 2003;140:93–100. DOI:10.1017/S0021859602002757
77 - Ferreira LMM, Garcia U, Rodrigues MAM, Celaya R, Dias-da-Silva A, Osoro K. The application of the n-alkane technique for estimating the composition of diets consumed by equines and cattle feeding on upland vegetation communities. Anim. Feed Sci. Technol. 2007;138:47–60. DOI: 10.1016/j.anifeedsci.2006.11.007
78 - Dove H, Mayes RW. Wild and Domestic Herbivore Diet Characterization. Satellite Meeting of the VI International Symposium on the Nutrition of Herbivores; 19–24 October 2003; Mérida, Yucatán, México. 2003. 88 p.
79 - Dove H, Moore AD. Using a least-squares optimization procedure to estimate botanical composition based on the alkanes of plant cuticular wax. Aust. J. Agric. Res. 1995;46:1535–1544. DOI:10.1071/AR9951535
80 - Newman JA, Thompson WA, Penning PD, Mayes RW. Least-squares estimation of diet composition from n-alkanes in herbage and faeces using matrix mathematics. Aust. J. Agric. Res. 1995;46:793–805. DOI:10.1071/AR9950793
81 - Martins H, Elston DA, Mayes RW, Milne JA. Assessment of the use of n-alkanes as markers to describe the complex diets of herbivores. J. Agric. Sci. 2002;138;425–434. DOI:10.1017/S0021859602002046
82 - Oliván M, Ferreira LMM, Celaya R, Osoro K. Accuracy of the n-alkane technique for intake estimates in beef cattle using different sampling times and feeding levels. Livest. Sci. 2007;106:28–40. DOI:10.1016/j.livsci.2006.06.015
83 - Charmley E, Dove H. Using plant wax markers to estimate diet composition and intakes of mixed forages in sheep by feeding a known amount of alkane-labelled supplement. Aust. J. Agric. Res. 2007;58:1215–1225. DOI: 10.1071/AR07187
84 - Azevedo EB, Poli CHEC, David DB, Amaral GA, Fonseca L, Carvalho PCF, Fischer V, Morris ST. Use of faecal components as markers to estimate intake and digestibility of grazing sheep. Livest. Sci. 2014;165:42–50. DOI:10.1016/j.livsci.2014.04.018
85 - Bezabih M, Pellikaan WF, Tolera A, Hendriks WH. Estimation of feed intake and digestibility in cattle consuming low-quality tropical roughage diets using molasses-based n-alkane boluses. Anim. Feed Sci. Technol. 2012;177:161–171. DOI: 10.1016/j.anifeedsci.2012.08.014
86 - Mayes RW, Lamb CS, Colgrove PM. The use of dosed and herbage n-alkanes as markers for the determination of herbage intake. J. Agric. Sci. 1986;107:161–170. DOI:10.1017/S0021859600066910
87 - Hendricksen RE, Reich MM, Roberton RF, Reid DJ, Gazzola C, Rideout JA, Hill RA. Estimating the voluntary intake and digestibility of buffel-grass and lucerne hays offered to Brahman-cross cattle using n-alkanes. Anim. Sci. 2002;74:567–577.
88 - Elwert C, Dove H. Estimation of roughage intake in sheep using a known daily intake of a labelled supplement. Anim. Sci. 2005;81:47–56. DOI:10.1079/ASC41940047
89 - Narvaez N, Brosh A, Pittroff W. Use of n-alkanes to estimate seasonal diet composition and intake of sheep and goats grazing in California chaparral. Small Rumin. Res. 2012;104:129–138. DOI:10.1016/j.smallrumres.2011.10.002
90 - Ferreira LMM, Oliván M, Rodrigues MAM, Osoro K, Dove H, Dias-da-Silva A. Estimation of feed intake by cattle using controlled-release capsules containing n-alkanes or chromium sesquioxide. J. Agric. Sci. 2004;142:225–234. DOI:10.1017/S0021859604004320
91 - Ferreira LMM, Garcia U, Rodrigues MAM, Celaya R, Dias-da-Silva A, Osoro K. Estimation of feed intake and apparent digestibility of equines and cattle grazing on heathland vegetation communities using the n-alkane markers. Livest. Sci. 2007;110:46–56. DOI:10.1016/j.livsci.2006.09.026
92 - Buschhaus C, Herz H, Jette R. Chemical composition of the epicuticular and intracuticular wax layers on adaxial sides of Rosa canina leaves. Ann. Bot. (Lond.) 2007;100:1557–1564. DOI: 10.1093/aob/mcm255
93 - Elwert C, Dove H, Rodehutscord M. Faecal alkane recoveries from multi-component diets and effects on estimates of diet composition in sheep. Animal 2008;2:125–134. DOI:10.1017/S1751731107000900
94 - Ferreira LMM, Celaya R, García U, Rodrigues MAM, Osoro K. Differences between domestic herbivores species in alkane faecal recoveries and the accuracy of subsequent estimates of diet composition. Anim. Feed Sci. Technol. 2009;151:128–142. DOI: 10.1016/j.anifeedsci.2008.11.003
95 - Hendricksen RE, Gazzola C, Reich MM, Roberton RF, Reid DJ, Hill RA. Using molasses as an alternative to controlled release devices for administering n-alkane markers to cattle. Anim. Sci. 2003;76:471–480.
96 - Elwert C, Kluth H, Rodehutscord M. Effect of variable intake of alfalfa and wheat on faecal alkane recoveries and estimates of roughage intake in sheep. J. Agric. Sci. 2004;14:213–223. DOI:10.1017/S0021859604004150
97 - Smith DG, Mayes RW, Hollands T, Cuddeford D, Yule HH, Malo Ladrero CM, Gillen E. Validating the alkane pair technique to estimate dry matter intake in equids. J. Agric. Sci. 2007;145:273–281. DOI:10.1017/S0021859607006788
98 - Dove H, Mayes RW, Lamb CS, Ellis KJ. Factors influencing the release rate of alkanes from an intra-ruminal, controlled-release device, and the resultant accuracy of intake estimation in sheep. Aust. J. Agric. Res. 2002;53:681–696. DOI:10.1071/AR01175
99 - Ordakowski AL, Kronfeld DS, Holland JL, Hargreaves BJ, Gay LS, Harris PA, Dove H, Sklan D. Alkanes as internal markers to estimate digestibility of hay or hay plus concentrate diets in horses. J. Anim. Sci. 2001;79:1516–1522. DOI:/2001.7961516x
100 - Stevens DM, van Ryssen JBJ, Marais JP. The use of n-alkane markers to estimate the intake and apparent digestibility of ryegrass and Kikuyu by horses. S. Afr. J. Anim. Sci. 2002;32:50–56. DOI:10.4314/sajas.v32i1.3791
101 - Peiretti PG, Meineri G, Miraglia N, Mucciarelli M, Bergero D. Intake and apparent digestibility of hay or hay plus concentrate diets determined in horses by the total collection of feces and n-alkanes as internal markers. Livest. Sci. 2006;100:189–194. DOI:10.1016/j.livprodsci.2005.08.016
102 - López López C, Celaya R, Rodrigues MAM, Osoro K, Ferreira LMM. Combination of long-chain alcohols and fatty acids with alkanes as faecal markers to estimate feed intake and digestibility of horses and cattle fed on grass-heathland vegetation communities. Can. J. Anim. Sci. 2016;96:221–231. DOI:10.1139/cjas-2015-0071
103 - Wilson H, Sinclair AG, DeB Hovell FD, Mayes RW, Edwards SA. Validation of the n-alkane technique for measuring herbage intake in sows. In: Proceedings of the British Society of Animal Science; March 1999; Penicuik, Midlothian, UK: BSAS; 1999. p. 177.
104 - Sehested J, Breinhild KK, Søegaard K, Vognsen L, Hansen HH, Fernández JA, Danielsen V, Kristensen VF. Use of n-alkanes to estimate grass intake and digestibility in sows. In: Dove H, Coleman SW, editors. Nutritional Ecology of Herbivores. Satellite Symposium: Emerging Techniques for Studying the Nutrition of Free Ranging Herbivores. San Antonio, TX (CD-ROM)
105 - Hulbert IA, Iason GR, Mayes RW. The flexibility of an intermediate feeder: Dietary selection by mountain hares measured using faecal n-alkanes. Oecologia 2001;129:197–205. DOI:10.1007/s004420100725
106 - Hatt JM, Mayes RW, Clauss C, Lechner-Doll M. Use of artificially applied n-alkanes as markers for the estimation of digestibility, food selection and intake in pigeons (Columba livia). Anim. Feed Sci. Technol. 2001;94:65–76. DOI: 10.1016/S0377-8401(01)00294-2
107 - Mayes RW, Lamb CS, Colgrove PM. Digestion and metabolism of dosed even-chain and herbage odd-chain n-alkanes in sheep. In: Proceedings of the 12th General Meeting of the European Grassland Federation; 4–7 July 1988; Dublin: EGF; 1988. pp. 159–163.
108 - Ohajuruka OA, Palmquist DL. Evaluation of n-alkanes as digesta markers in dairy cows. J. Anim. Sci. 1991;69:1726–1732. DOI:/1991.6941726x
109 - Gudmundsson O, Thohallsdottir AG. Evaluation of n-alkanes for intake and digestibility determination in horses. In: Gibb MJ, editor. Proceedings of the IXth European Intake Workshop; 18–20 November 1998; North Wyke, Devon, UK: IGER; 1998. pp. 1–4
110 - O’Keefe NM, McMeniman NP. The recovery of natural and dosed n-alkanes from the horse. Anim. Prod. Aust. 1998;22:337. Available from:'Keefe98a.PDF [Accessed: 2016-07-12]
111 - Vouzela CFM. Validation of n-alkanes as markers in herbivore [thesis]. Angra do Heroísmo: University of Azores; 2002.
112 - Elwert C, Dove H, Rodehutscord M. Effect of roughage species consumed on faecal alkane recovery in sheep, and effect of sample drying treatment on alkane concentrations. Aust. J. Exp. Agric. 2006;46:771–776. DOI:10.1071/EA05303
113 - Himmelsbach DS. Structure of forage cell walls-session synopsis. In: Jung HG, Buxton DR, Hatfield RD, Ralph J, editors. Forage Cell Wall Structure and Digestibility. Madison, WI: ASA-CSA-SSSA; 1993. pp. 271–283. DOI: 10.2134/1993.foragecellwall.c11
114 - Kelman W, Bugalho M, Dove H. Cuticular wax alkanes and alcohols used as markers to estimate diet composition of sheep (Ovis aries). Biochem. Syst. Ecol. 2003;31:919–927. DOI:10.1016/S0305-1978(03)00081-4
115 - Vargas-Jurado N, Tanner AE, Blevins SR, McNair HM, Mayes RW, Lewis RM. Long-chain alcohols did not improve predictions of the composition of tall fescue and red clover mixtures over n-alkanes alone. Grass Forage Sci. 2015;70:499–506. DOI:10.1111/gfs.12134
116 - Ferreira LMM, Oliván M, Celaya R, Garcia U, Rodrigues MAM, Osoro K. The use of n-alkanes to estimate diet composition of ruminants grazing on species diverse plant communities—Effect of feeding selectivity on diet composition estimates. Livest. Sci. 2007;111:114–123. DOI: 10.1016/j.livsci.2006.12.008