browsing sheep and goats

Small Ruminant Research 59 (2005) 123–139

Review article

Using n-alkanes and other plant wax components to estimate intake, digestibility and diet composition of grazing/browsing sheep and goats夽 H. Dove a,∗ , R.W. Mayes b a

CSIRO Plant Industry, G.P.O. Box 1600, Canberra, A.C.T. 2601, Australia b The Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, UK

Abstract The use of plant-wax markers for estimating forage intake, diet composition and supplement intake in grazing/browsing sheep and goats is reviewed in relation to some of the shortcomings of pre-existing techniques. The saturated hydrocarbons (alkanes) of plant wax are well validated as markers for estimating intake. Alkane-based estimates of intake have the advantage of accommodating individual differences in digestibility and those arising from supplement/forage interactions. Alkanes can also provide an estimate of diet composition and hence the inputs required to estimate herbage intake. Total intake can thus be partitioned into intake of individual species. It is probable that the alkane approach to estimating diet composition can discriminate fewer species in the diet of sheep and especially goats, than these animals encounter in complex plant communities. We discuss approaches to coping with this issue, such as grouping species in diet composition estimates, and the use of other plant-wax markers as diet composition markers. The long-chain alcohols and fatty acids are shown to have particular promise for discriminating a greater number of species in the diet. We then discuss the estimation of supplement intake using plant hydrocarbons, as a special case of the estimation of diet composition. Finally, we demonstrate that when supplement intakes are known, alkane-based estimates of the supplement proportion in the diet can be used to estimate the intake of all dietary components, without the need to dose animals with synthetic alkanes. Crown Copyright © 2004 Published by Elsevier B.V. All rights reserved. Keywords: Intake; Diet composition; Sheep; Goats; Alkanes; Plant wax

1. Introduction 夽 This paper is part of the special issue entitled: Methodology, nutri-

tion and products quality in grazing sheep and goats, Guest Edited by P. Morand-Fehr, H. Ben Salem and T.G. Papachristou. ∗ Corresponding author. E-mail address: [email protected] (H. Dove).

Sheep and goats frequently consume a diet which differs in terms of plant species, plant parts and nutrient content from the average of the available plant biomass. An accurate estimate of their nutrient intake requires an estimate of both the botanical composition of the diet

0921-4488/$ – see front matter. Crown Copyright © 2004 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2005.05.016

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(or at least a sample of the ‘consumed diet’) and of total intake. However, these measurements are difficult to make under field conditions because of shortcomings in the current methods of estimation. Techniques for estimating diet composition and intake have been extensively reviewed (Langlands, 1987; Dove and Mayes, 1996; Mayes and Dove, 2000) and can be based either on measurements of the plant biomass or on animalbased measurements. In this review, we will not discuss the former. Rather, we will consider selected animalbased techniques, especially marker-based techniques, used to estimate diet selection and intake and highlight some of the problems associated with their use. We will then discuss the possible use of the components in plant cuticular wax as markers for estimating diet composition, intake and digestibility in sheep and goats.

2. Estimation of intake In free-ranging ruminants, intake has been estimated from behavioural data such as grazing time and bite size (e.g. Decandia et al., 2000), and from techniques involving the turnover of water or sodium (Silanikove et al., 1987). However, over the last 50 years, the most successful and widely used methods for estimating intake have taken advantage of the relationship between intake (I), the digestibility of the whole diet (D) and the resultant faecal output (F), that is: I=

F 1−D

(1)

Intake is estimated from separate estimates of faecal output and the digestibility of the consumed diet. There are errors attendant to the estimation of both F and D, but the latter measurement is the greater cause for concern (Langlands, 1987; Dove and Mayes, 1996). 2.1. Estimating faecal output Total faecal collection under field conditions is labourious and can disturb normal foraging behaviour. Faecal output is therefore more commonly estimated from the faecal dilution of a so-called ‘external’ marker, administered orally. A wide range of external markers has been used for the estimation of faecal output (see Table 2 in Mayes and Dove, 2000) but to date, none has met the criteria required of an ideal marker (Kotb

and Luckey, 1972). However, chromium sesquioxide (Cr2 O3 ) has been shown to have a high faecal recovery and to perform satisfactorily as a faecal output marker in both sheep (see Langlands, 1987; Mayes and Dove, 2000) and goats (e.g., Kababya et al., 1998). It is administered orally in gelatin capsules, paper pellets or by using an intra-ruminal, controlled-release device (CRD). Once faecal Cr concentrations have equilibrated, faecal output is computed from Cr dilution in faeces. Possible errors in the use of Cr2 O3 for faecal output estimation have been reviewed by Langlands (1987), Parker et al. (1990), Dove and Mayes (1991) and Dove et al. (2000). The use of a CRD to administer Cr helps to overcome some of the problems associated with cyclic changes in faecal Cr concentration arising from once- or twice-daily dosing. However, even if faecal output were to be estimated with little error, a greater cause for concern is with the estimate of digestibility. 2.2. Estimating the digestibility of the diet Errors in the estimate of digestibility can seriously reduce the accuracy of the estimate of intake derived using Eq. (1), especially when diet digestibility is high and thus the denominator of the equation is small (Langlands, 1987; Dove and Mayes, 1996). A major difficulty in obtaining an accurate estimate of digestibility is that of obtaining a sample representative of the diet actually consumed by the grazing or browsing animal. 2.2.1. In vitro estimates of digestibility Animals fistulated at the oesophagus can be used to obtain samples ‘representing’ the diet of the test animals. In vitro estimates of the digestibility of these samples can then be conducted (e.g., Jones and Hayward, 1975). These techniques must be calibrated against in vivo estimates of digestibility. The possible problems associated with this approach have been discussed in detail elsewhere (Langlands, 1987; Dove et al., 2000; Mayes and Dove, 2000). A major issue is that it is assumed that the samples obtained by the oesophageal-fistulated animals represent the diet of the intact, test animals. There are doubtless occasions when this is so (e.g., Dove, 1998; Dove et al., 2000), but the assumption is frequently questioned because oesophageal extrusa samples are

H. Dove, R.W. Mayes / Small Ruminant Research 59 (2005) 123–139

usually collected over a time-span of minutes, whereas the test animals may be grazing or browsing the area for days or weeks. Moreover, the diet selected by the oesophageal-fistulated animals may differ from that of the test animals because the former animals are surgically prepared, are of different sex or physiological state, or are handled and managed differently from the test animals. These points were discussed in more detail by Mayes and Dove (2000), who pointed out that there was no absolute way to test the assumptions. The possible problems associated with the short collection period used to obtain oesophageal extrusa could perhaps be overcome by the use of a cannula fitted with a remotecontrol valve (Raats et al., 1996). In foraging goats, this permitted the collection of a diet sample over the course of a whole day, without interrupting normal grazing behaviour. A further problem is that the results of in vitro digestibility assays are often applied to animals which may differ markedly in sex, stage of growth, reproductive status, intake level and even species from the animals used in the original in vitro/in vivo calibration. In addition, the single in vitro estimate of digestibility is applied to all the test animals, despite the strong likelihood that digestibility of a given diet differs among individual animals. As we will indicate below, one of the major advantages of the n-alkane approach to the estimation of intake is that differences in digestibility between individuals can be accommodated more adequately, especially if intake is estimated following an estimate of diet composition in individuals. Finally, grazing animals are often fed supplements when pasture quantity or quality is inadequate. The in vitro methods cannot accommodate possible interactions between forage and supplement during digestion e.g., the possible reduction in the digestibility of roughages when animals are also consuming starchy components such as cereal grain. We stress that these are potential rather than inevitable sources of error in estimating intake, and that accurate data of great scientific and practical importance have been obtained using the Cr/in vitro procedure. Of the three problems listed, the first is probably the greatest cause for concern, because it is the one over which the researcher has least control. A possible advantage of the use of plant alkanes as intake markers is that they can provide a check of the assumption

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that fistulated and test animals are consuming the same thing (see below and Dove et al., 2000). 2.2.2. Digestibility based on ‘internal markers’ To try to overcome some of the problems associated with in vitro estimates of digestibility, indigestible diet components have been used as indicators or ‘internal markers’ of digestibility. In individual animals, the increase in marker concentration between diet and faeces provides an estimate of the indigestibility of the diet i.e., the proportion of consumed diet which is excreted in faeces, calculated as (marker concentration in feed)/(marker concentration in faeces). Digestibility is then calculated as (1 − indigestibility). An advantage of the internal marker approach is that it can be applied to animals consuming a mixture of forage and supplement and will reflect the interactions occurring between these components during digestion. This approach requires that the intake and internal marker concentration of the supplement must be known. Mayes and Dove (2000) have recently reviewed the range of dietary components which have been used as internal markers (see their Table 3). The approach has had variable success, mainly because many of the proposed markers fall well short of the criteria required for an ‘ideal marker’ (Kotb and Luckey, 1972). In particular, they are often not chemically discrete compounds with specific assays (e.g. ‘chromogen’, potentially indigestible cellulose), so there is uncertainty about whether the marker assayed in faeces is chemically identical to that assayed in the feed. This has probably contributed to the variable results used with these empirical internal markers.

3. Using plant cuticular wax markers in studies of the intake of herbivores 3.1. The nature of plant cuticular wax The wax on the external surface of plants (cuticular or epicuticular wax) is a complex mixture of aliphatic lipid compounds, the chemical composition of which differs greatly among different plant species and, to a lesser extent, between different parts of the plant. Leaves and floral parts of the plant tend to have the

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3.1.1. Hydrocarbons Hydrocarbons are present in the waxes of most higher plants, though only rarely as the main component; n-alkanes are the most common hydrocarbons. They are present as mixtures with chain lengths ranging from 21 to 37 carbon atoms. Over 90% of n-alkanes have odd-numbers of carbon atoms, with C29 , C31 and C33 alkanes being dominant in most pasture species (Table 1). The unsaturated aliphatic hydrocarbons (alkenes) are relatively common in the floral parts of plants and also in the leaves of a number of tree and shrub species. Most of the alkenes are odd-chain monoenes, with chain lengths ranging from 23 to 33 carbon atoms. Alkenes have potential as diet composition

highest concentrations of wax (Tulloch, 1976; Dove and Mayes, 1991, 1996). The composition of plant waxes has been comprehensively reviewed elsewhere (Tulloch, 1976; Dove and Mayes, 1991). In the present paper, we will restrict discussion to the main plant wax compounds which have been used in or offer potential for studies of herbivore nutrition. The initial suggestion was that the long-chain fatty acids (LCFA) of plant wax might be useful markers (Grace and Body, 1981), but because of their relative inertness and ease of analysis, n-alkanes were the first plant-wax compounds to be used as potential faecal markers (Mayes and Lamb, 1984; Mayes et al., 1986) and have since been the most frequently used wax marker in herbivore nutrition studies.

Table 1 Concentrations (mg/kg DM) of the major (odd-chain) n-alkanes and the major long-chain alcohols (even-chain) in a selected group of dicotyledons and monocotyledons n-Alkanes

Long-chain alcohols

C25

C27

C29

C31

C33

C35

Dicotyledons Brassica oleracea Calluna vulgaris Fagus sylvatica Lotus corniculatus cv. Goldie Lotus pedunculatus cv. Maku Picea sitchensis Pinus sylvestris Trifolium glomeratum Trifolium repens Trifolium subterraneum Trifolium striatum

1-C24 -OH

1-C26 -OH

10-C29 -OH

1-C28 -OH

1-C30 -OH

2 13 18 14 15 2 13 11 9 4 10

6 63 361 38 151 3 28 35 35 16 48

456 160 13 38 212 8 21 313 108 250 990

136 636 3 34 55 4 1 267 124 74 68

5 458 3 33 37 2 10 36 15 10 8

0 14 0 0 1 0 4 0 4 0

23 363 191 13 28 85 81 48 18 193 37

142 167 89 2154 2463 18 58 124 143 408 214

196 14 7 0 0 2065 1853 0 0 0 0

25 260 3 905 1327 14 13 73 61 281 444

29 298 39 1015 1285 20 114 1199 1297 4709 1259

Monocotyledons Austrodanthonia racemosa Austrodanthonia richardsonii Bothriochloa macra Bromus catharticus Chloris gayana Cynodon dactylon Digitaria dactyla Festuca arundinacea Lolium perenne Microlaena stipoides Paspalum dilatatum Paspalum notatum Pennisetum clandestinum Phalaris aquatica Setaria anceps Themeda triandra Vulpia myuros

8 19 22 6 12 0 5 24 10 5 0 0 0 27 32 7 28

17 26 132 15 89 11 24 42 33 12 8 0 7 17 82 20 41

73 58 65 116 180 30 55 129 77 70 12 5 12 21 62 59 176

613 90 111 60 243 66 96 216 103 216 56 35 79 16 74 278 184

625 11 34 34 137 91 126 59 84 156 36 168 195 7 25 255 37

13 0 4 4 32 58 42 2 11 8 13 308 204 2 5 41 3

45 66 61 13 64 31 88 27 104 11 41 31 30 19 28 26 80

66 435 44 85 56 25 76 639 2628 17 15 9 9 3726 20 33 901

– 0 – 17 – – – 10 10 – – – – 23 – – 13

126 171 285 4052 187 – – 101 446 35 – – – 45 – 15 42

455 131 221 84 149 0 0 58 627 1660 20 0 0 472 24 69 65

Adapted in part from data in Bugalho et al. (2004) and Ali et al. (2005).

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markers (see below), despite their relatively low faecal recoveries. 3.1.2. Wax esters and free fatty acids and alcohols In most plants, wax esters of saturated, unbranched LCFA and long-chain alcohols (LCOH) are the main component of plant wax, with chain lengths in the range C32 –C64 . Although plant-wax LCFA have been considered as potential digestibility and diet composition markers (Grace and Body, 1981; Ali et al., 2004, 2005) and the LCOH have been evaluated as diet composition markers (Kelman et al., 2003; Bugalho et al., 2004; Ali et al., 2005), it should be noted that these assessments have been based upon their total concentrations (free plus esterified), since the analytical methods involve a saponification step which cleaves the wax esters. As yet, neither the intact wax esters or the free LCFA or LCOH of plant wax have been evaluated as markers. The LCFA of plant wax are predominantly mixtures of straight-chain, saturated compounds with evennumbered chain lengths up to C34 . In nutritional studies, LCFA with chain lengths C20 –C34 have been used because they have high faecal recoveries (Grace and Body, 1981; Ali et al., 2004) and because, unlike some shorter LCFA, they derive exclusively from plant material. Most of the LCOH are primary alcohols having the same range in even-numbered chain length as the LCFA. Whilst the concentrations of primary odd-chain alcohols are negligible, secondary odd-chain alcohols can occur at high levels in certain plants; for example, many conifers contain high concentrations of the secondary alcohol, 10-nonacosanol (C29 ). The wide variation in alcohol patterns in different plants (Table 1), combined with relatively high faecal recoveries makes the LCOH good diet composition markers (see below). There is also potential to use them as intake markers. The LCFA also show promise as potential diet composition markers. 3.2. Using n-alkanes to estimate intake The saturated hydrocarbons were initially suggested as digestibility markers (Mayes and Lamb, 1984), but are not ideal for this purpose since they have faecal recoveries of less than 100%. The conceptual leap which allowed their use as intake markers was the realization by Mayes et al. (1986) that if ani-

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mals are dosed orally with a synthetic even-chain alkane of similar recovery to a plant alkane of adjacent carbon-chain length, then the faecal recovery of the markers is no longer an issue. In essence, the plant alkane is functioning as an internal marker to provide an estimate of digestibility, whilst the dosed alkane functions as the external, faecal output marker. However, in practice, digestibility and faecal output are not calculated separately but rather, for a given alkane pair (plant odd-chain alkane i and dosed evenchain alkane j), intake (I) is calculated directly from herbage alkane concentrations (Hi and Hj ), faecal concentrations (Fi and Fj ), faecal recoveries (Ri and Rj ) and the dose rate of alkane j, using the following equation: I =
F

j ×Ri

Fi ×Rj



Dose ratej × (Hi − herbage contentj )

(2)

The derivation of a slight variant of this equation from Eq. (1) above is given by Dove and Mayes (1991; see their Appendix 1). Note that it is ultimately the ratio of the faecal alkane concentrations which is important, not their absolute concentrations. Furthermore, if the faecal recoveries of the plant alkane (Ri ) and the dosed alkane (Rj ) are equal, errors associated with incomplete faecal recovery cancel out and an unbiased estimate of intake will be obtained. This is in contrast to methods based on the separate use of internal and external markers to estimate digestibility and faecal output separately. Note also that it does not matter if the herbage also contains the dosed alkane; this is allowed for in the denominator of Eq. (2). The work of Mayes et al. (1986) and subsequent studies (reviewed by Dove and Mayes, 1996) have demonstrated that the faecal recovery of longer alkanes adjacent in chain length is very similar (see below) so that intake can be estimated accurately using, for example, dosed C32 alkane and either C31 or C33 alkane from the plant. In practice, it is therefore assumed that the recoveries of the herbage and dosed alkanes are equal, and recoveries are not used explicitly in the estimation of intake using Eq. (2). Note that intake could equally well be computed using a plant LCOH or LCFA, together with a dosed alkane, provided the assumption of similar faecal recovery of dosed and natural marker was met.

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The alkane method for estimating intake offers a number of advantages over other techniques. 1. Provided that the sample of vegetation analysed for alkanes is representative of the actual diet consumed, the method gives intakes estimates which can be regarded as ‘individual’, to the extent that they accommodate the digestibility of the diet in individual animals. Intake estimates are even more truly ‘individual’ if the composition of the diet consumed by individual animals can also be computed using alkanes (see below). 2. If, in addition to being dosed with an even-chain alkane to allow the estimation of intake, the animals are also dosed with an external marker, faecal output can be estimated and from this, whole-diet digestibility. The marker Cr2 O3 could be used but hexatriacontane (C36 alkane) has been shown to have a consistently high faecal recovery of ∼95% and can be used instead of Cr2 O3 . 3. The method can be extended to the simultaneous estimation of diet composition, so that the intake of individual plant species or plant parts can be estimated (see below). 4. It can be used with animals which are also receiving supplementary feeds, provided the alkane concentrations of the supplement and supplement intake are known. Methods for estimating supplement intake are discussed later in this review. 5. The plant, faecal and external marker alkane concentrations are determined at the same time by the same analytical procedure, which reduces analytical

error and bias. Since it is the ratio of the concentrations of alkanes in faeces which is used, it is not even necessary to obtain absolute faecal concentrations. 6. It can be calculated from Eq. (2) that differences in faecal recovery between the alkane pair used to estimate intake (i.e. recoveryi cf. recoveryj ) will result in a linear and almost quantitative error in the estimate of intake, as discussed earlier (Dove and Mayes, 1996). A difference in faecal recovery of, say 3%, will thus generate an error of 3% in estimated intake. Moreover, this error is independent of the level of digestibility of the diet. By contrast, it can be calculated from Eq. (1) that an under-estimate of three percentage units in the in vitro digestibility will result in an over-estimate of intake of 6.3% if digestibility is 50% and almost 18% if digestibility is 80%. Indoor validation studies have shown that the alkane procedure provides reliable estimates of measured intake in sheep and, in a single trial, in goats (Table 2), across a wide range of forage types, including browse. There is no evidence to date that plant secondary compounds cause any interference to alkane-based estimates of intake. By definition, validation under grazing or browsing conditions is not possible, because ‘actual’ intakes are unknown and because alternative techniques for estimating intake may be no better and possibly inferior. The alkane and Cr-in vitro methods for estimating intake have been compared in sheep (Piasentier et al., 1995; Dove et al., 2000). Piasentier et al. (1995)

Table 2 Comparison of measured herbage intakes of sheep and goats with those estimated using dosed and herbage alkanes Source

Animals/conditions

Known intake (g DM/day)

Mean discrepancy ± S.E. (g DM/day)a

Sheep Mayes et al. (1986) Vulich et al. (1991) Dove and Oliv´an (1998) Dove et al. (2002a) Sibbald et al. (2000) Lewis et al. (2003) Valiente et al. (2003)

36 kg lambs/fresh herbage 34 kg lambs/fresh herbage 30 kg sheep/chaff + sunflower meal Adult sheep/pre-frozen herbage Adult sheep (group 1)/pelletted grass meal Lambs (0.3, 0.45 of mature weight; forage diets) 57 kg ewes/straw + barley grain

579 778 720 914 2040 2016 748

0 −20 −6 0.2 −39 −49 −46

Goats Gir´aldez et al. (2004)

43 kg goats/fresh herbage, pulse dose of alkane

808

a b

± ± ± ± ± ± ±

4.7 (0%) 9.3 (2.6%) 6.4 (0.8%) 15.5 (−0.02%) 27.4 (1.9%) 28.3 (2.4%)b 24.0 (6.1%)

18 g/day (−2.1%)

Calculated as (known intake − estimated intake). Values in parenthesis are the percentage over- or under-estimates of known intake. Calculated from the data in their Table 3.

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noted that in both housed and grazing sheep, intakes estimated using the Cr-in vitro method were always higher than those estimated using the C31 :C32 alkane pair, especially at higher levels of intake. In grazing ewes, Dove et al. (2000) found that the relationship between the two methods for estimating herbage intake was affected both by the physiological state of the animals (late pregnancy versus early lactation versus mid lactation) and by the level of intake. The major cause of the discrepancy between the intake estimates was that in vitro estimates of digestibility did not adequately represent in vivo digestibility. Their results indicate that in contrast to the in vitro procedure, the alkane procedure accommodated the real differences in digestibility between animals. They also indicate clearly that there is no simple relationship between estimates of herbage intake based on these different methods. A major consideration in using the alkane method is that the procedures used for dosing of the even-chain alkane do not generate cyclic temporal variation in the faecal alkane ratio used in the intake calculation. Evenchain alkanes have now been administered to sheep or goats using a wide range of carrier matrices, including shredded paper (Mayes et al., 1986; Gir´aldez et al., 2004), cellulose powder in gelatin capsules (Dove et al., 2000; Gir´aldez et al., 2004), paper bungs or filter tips (Lewis et al., 2003; Gir´aldez et al., 2004) and xanthan gum suspension (Marais et al., 1996). An intra-ruminal controlled-release device (CRD) has also been tested (Molle et al., 1998; Dove et al., 2002a; Ferreira et al., 2004) and commercially released (Captec AlkaneTM , Captec (NZ), Auckland, New Zealand). These devices greatly reduce labour inputs and are designed to deliver alkane over a period of 20 days after insertion into the rumen; faecal sampling can commence 5–7 days after insertion. They have been shown to give accurate estimates of intake in sheep (see Table 2 and Molle et al., 1998; Dove et al., 2002a). In situations where very high intakes might be expected (e.g., during lactation), two devices can be inserted into the rumen without compromising daily release rates (Dove et al., 2002a). Moreover, there appears to be no effect of feeding level or feeding frequency on alkane release rate, at least under the conditions tested to date (Dove et al., 2002a). Under grazing conditions, the alkane CRD is a convenient mode of alkane delivery, but care must be taken to establish the exact release rate under the specific conditions of the experiment (Ferreira et al., 2004). These

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can be obtained either by monitoring CRD release in rumen fistulated animals (see Dove et al., 2002a; Ferreira et al., 2004) or by taking rectal grab samples to establish the time when alkane release ceases. If absolute estimates of intake are required, the release rate provided by the manufacturer should be taken only as a guide. However, it may be adequate to provide estimates of comparative intakes of different treatments. In addition to containing C32 alkane for the estimation of intake, the commercial alkane CRD also contains C36 alkane as a faecal output marker which thus also allows an estimate of the in vivo digestibility of the diet consumed by the grazing animal (Decandia et al., 2000).

4. Estimation of diet composition Animal-based methods for estimating the species composition of the diet have included: the microhistological examination of plant fragments (usually cuticle) in oesophageal extrusa, a gut compartment or in faeces; the estimation of carbon-isotope ratios in such samples or in wool or hair, and; the use of plant-wax components as markers. The merits of these various methods have been reviewed extensively (Holechek et al., 1982; Dove and Mayes, 1996; Mayes and Dove, 2000) and in the present paper, we will discuss the first two approaches only briefly. 4.1. The use of oesophageal-fistulated animals to estimate diet composition In domestic herbivores at least, oesophagealfistulated animals have also been used to estimate the composition of the diet in terms of plant species (e.g., Salt et al., 1994; Dove, 1998) or parts (e.g., Leury et al., 1999). Several authors have presented data to indicate that fistulated and test animals have selected similar diets (e.g., Dove, 1998; Leury et al., 1999; Dove et al., 2000) but this cannot always be assumed to be so. Prior to the use of alkanes as diet composition markers, there was no way to test this assumption. However, it can now be argued that if fistulated and test animals are consuming the same diet, then their faecal alkane profiles should be similar, or there should be similarity between the alkane profile of oesophageal extrusa collected by the fistulated animals and the faeces of the test animals

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(after correction to 100% faecal alkane recovery—see below). Dove et al. (2000) adopted the latter approach to investigate whether oesophageal samples collected by castrate male sheep could be assumed to reflect the diet of grazing ewes. When alkane profiles were expressed as proportions of total alkane concentration, there was very close correspondence between the alkane profiles of oesophageal samples from fistulated animals and faeces samples from either pregnant or lactating ewes. This suggests that they were selecting similar diets. Similarities in the diet of fistulated and test animals were observed by Dove et al. (1999) and Leury et al. (1999) in sheep grazing senescing pasture. 4.2. Microhistological procedures The many variants of this approach all depend upon the identification of plant tissue fragments in prepared samples of oesophageal extrusa, stomach contents or faeces (Holechek et al., 1982). Diet composition is then estimated in terms of the proportion of these fragments coming from each plant species. In order to relate diet composition and intake, calibrations will usually be needed to convert these proportions to an estimate of diet composition on a dry matter basis. The method has been used extensively with wild animals, often using stomach contents from slaughtered animals (Holechek et al., 1982). Faecal sampling is more generally applicable and allows repeat sampling and larger numbers of animals to be used, but also has major disadvantages, principally the effect of differential digestion of fragments arising from the different plant species. There is also the problem that a large proportion of fragments can remain unidentifiable, which reduces the quantitative reliability of the method. Nevertheless, the approach can be very useful for establishing the presence or absence of a particular plant species in the diet and can thus complement other methods such as the use of plant wax markers, by ensuring that particular species are included in the estimate of diet composition. 4.3. Stable isotope discrimination Tissue from plants which exhibit the C3 or C4 pathways of photosynthesis contains different ratios of the 13 C and 12 C isotopes of carbon. This difference (␦13 C) can be used to estimate the proportion of C3 and C4

herbage in the diet based on oesophageal (Coates et al., 1987) or faecal samples (Jones et al., 1979). This is especially useful in tropical grazing systems, in which grass species make up the main source of C4 plants whilst legumes, browse and forbs comprise mainly C3 plants. The method is of limited use in colder environments, where there are few C4 plants. The effect of differential digestion remains an issue. When faeces samples are used, the method will tend to underestimate the intake of plants of high digestibility, since less of their carbon remains in faeces. Moreover, the faecal carbon isotope ratio can be perturbed by faecal endogenous carbon. The method cannot resolve the diet to the species level, but is still useful in providing an estimate of legume content of the diet, a key variable in many management systems. 4.4. Using plant wax markers to estimate diet composition Since plant species differ in the alkane, the LCOH and the LCFA profiles of their cuticular wax, in theory any or all of these compounds could be used to estimate diet composition. However, because they are much easier to analyse in plant wax, only the alkanes have been widely used as diet composition markers (Mayes and Dove, 2000). A major advantage of using plant wax markers is that the diet composition is estimated in the actual test animals, so that the resultant alkane concentrations in their consumed diet can be calculated and used as inputs in Eq. (2) to estimate herbage intake (Mayes et al., 1986). This obviates the need to use oesophageal-fistulated animals to obtain a sample of ‘consumed diet’. In terms of scientific, labour and animal welfare considerations, this must be seen as a major advantage of the alkane approach. However, it also means that the accuracy of the intake estimate is itself a function of the accuracy of the diet composition estimate. Care must thus be exercised in estimating diet composition, or the estimate of herbage intake may also be incorrect. 4.4.1. Using n-alkanes as diet composition markers There are marked differences in the alkane profiles of plants, both in forage and in browse species (Table 1). These differences have been used to estimate the species composition of oesophageal extrusa (Salt et

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al., 1994; Dove, 1998) and have been validated and used as markers for estimating the diet of both sheep (e.g., Mayes et al., 1994; Dove and Mayes, 1996; Kelman et al., 2003; Lewis et al., 2003; Valiente et al., 2003) and goats (e.g., Merchant, 1996; Brosh et al., 2003). The use of alkanes (or any other plant wax marker) to estimate the composition of a mixture relies on the principle that the alkane profile in the mixture (be it oesophageal extrusa, digesta or faeces) must arise from some combination of the alkane profiles in the components which might contribute to that mixture (plant species and/or plant parts). There are a number of methods for making the necessary calculations to compare these profiles. However, we stress that it is much more important to ensure that the inputs to the calculations are as accurate as possible (see below), than to be concerned about which methods of calculation are used. If the number of marker alkanes at least equals the number of diet components, then in theory at least, diet composition could be calculated using simultaneous equations. However, this approach rapidly becomes computationally difficult with complex mixtures. Perhaps more importantly, when there are more alkanes present than there are dietary components, the use of simultaneous equations can involve an a priori and possibly arbitrary choice of which alkanes to use in the calculations. Moreover, the information provided by those markers which are not included is lost. In such situations, least-squares optimisation methods are more satisfactory, for which a number of similar algorithms have been used (e.g. Mayes et al., 1994; Dove and Moore, 1995; Newman et al., 1995). Basically, all these algorithms attempt to minimize the squared deviations between the observed alkane concentrations in faeces (or other mixtures) and the concentration profile arising from the estimate of diet composition, that is: 

[actual − estimated]2alc:1...n or :  [Fi − (xAi + yBi + zCi )]2alc:1...n

(3)

where, for n alkanes: Fi = actual faecal concentration of alkane i; x, y and z = respective amounts of dietary components A, B and C, Ai , Bi and Ci = respective concentrations of i in A, B and C. The amounts of dietary components A, B and C can be converted into dietary proportions of these compo-

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nents from expressions such as: Dietary proportion of component A =

x (4) x+y+z

Eq. (3) can be couched in terms of either concentrations or by expressing individual alkane concentrations in faeces and dietary components as proportions of their total (Mayes et al., 1994). However, the use of alkane concentrations confers a major advantage over optimisations based on alkane proportions. As dimensional analysis can demonstrate, x, y and z in the above equation are the amounts which, together, will result in one kilogram of faeces. This information can thus be used to obtain an estimate of whole-diet digestibility, given by: Whole-diet digestibility =

(x + y + z) − 1 x+y+z

(5)

This is not only useful information in its own right, but also serves as a check on whether the least-squares diet composition estimate is ‘realistic’. A similar approach, when one or more of the component intakes is known, is to use the optimisation routine to determine the actual faecal output and intakes of the remaining dietary components (Dillon et al., 2002): Estimated alkanei =

xAi + yBi + zCi fecal output

(6)

The main advantage of this approach is that intake estimates can be obtained without the need to dose animals (although dosed alkanes or natural waxes can be accommodated). An assumption inherent in these algorithms is that faecal alkane recovery is complete. The results of many studies have demonstrated that this is not so (Fig. 1). Faecal alkane recoveries show a curvilinear increase with alkane chain length in sheep. Brosh et al. (2003) recently reported a linear increase in odd-chain recovery in goats (Fig. 1); even-chain alkane recoveries were lower and unaffected by chain length. To obtain an unbiased estimate of diet composition from plant and faecal alkane concentrations, it is therefore important to adjust the latter for incomplete faecal recovery of alkanes. The required alkane recovery data can be obtained from metabolism cage studies or, in grazing animals, after dosing with various synthetic alkanes of different chain length (e.g., C24 , C28 , C32 , C36 ). The recoveries of other alkanes can then be calculated by interpolation

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Fig. 1. Effect of carbon-chain length on the faecal recovery of alkanes and alkenes of equivalent chain length. Alkane recovery data for sheep adapted from Dove and Oliv´an (1998;
), Elwert et al. (2004; ) and Elwert and Dove (2005; () mixed diets, () chaff diet). Goat alkane recoveries (♦) calculated from the regression for odd-chain alkanes in Brosh et al. (2003). Alkene recovery data (sheep only) adapted from Elwert et al. (2004; 䊉), Elwert and Dove (2005; ( ) mixed diets) and Dove and Oliv´an (2005; +).

(Dove and Mayes, 1996). The values presented in Fig. 1 suggest literature values could also be assumed, though this should be done with caution. It should be noted that recovery values can be computed either as absolute recoveries (proportion of known alkane intake excreted in faeces), or as relative recoveries (the recoveries of a suite of alkanes relative to an assumed value (usually 1) for the longest alkane). To obtain an unbiased estimate of whole-diet digestibility with least-squares minimisation calculations, absolute recovery values must be used. For an estimate of diet composition only, relative recoveries are sufficient. There have been a number of comparisons of the algorithms for estimating diet composition. Hameleers and Mayes (1998) compared three different leastsquares procedures for estimating diet composition using alkanes (Mayes et al., 1994; Dove and Moore, 1995; Newman et al., 1995) and found that they gave almost identical answers. The mathematical procedures available to estimate diet composition are thus satisfactory. It is more important to pay proper attention to faecal recovery corrections, and to the choice of which alkanes to use in the calculations.

The maximum number of components which can be discriminated in the diet is theoretically limited to the number of available alkanes (C21 –C37 ), thus making it theoretically possible to distinguish more than 15 dietary components. However, fewer dietary components can be discriminated, for two reasons. First, the low concentrations of the even-chain alkanes (often <10–30 mg/kg OM) mean that these are usually (but not always) less useful as markers. Second, it is likely that the reliability of the diet composition estimate will decline as the number of components increases, because there is an increased likelihood that different combinations of components could result in the same faecal alkane patterns. Despite this, field studies with goats and sheep, involving varying degrees of external validation, indicate that good estimates of the composition of multi-component diets can be obtained using alkanes (Mayes et al., 1994; Dove, 1998; Dove et al., 1999; Kelman et al., 2003). The data in Table 3 are recalculated from the results reported by Mayes et al. (1994) for the diet composition of lactating goats grazing a partially forested area in Norway, following radiocaesium deposition after the Chernobyl nuclear accident. Radiocaesium intakes calculated from the estimated diet composition and intake were compatible with the measured radiocaesium content of the milk produced by the goats. 4.4.2. Which alkanes to use in diet selection calculations? In some grazing situations (e.g., grass/clover pastures) there are more markers (alkanes) available than there are species in the diet. Although the above Table 3 Estimated diet composition in goats grazing a forested area near Griningsdalen, Norway, derived from the use of plant and faecal nalkanes as markers Diet composition Plant species

Proportion in diet

Betula pubescens Salix spp. Deschampia cespitosa Betula nana Carex spp. Melampyrum sylvaticum Juniperus communis

0.042 0.589 0.051 0.011 0.025 0.206 0.076

From Mayes et al. (1994).

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least-squares procedures can accommodate information about all available alkanes, it is possible that some alkanes may discriminate better between components. Conversely, other alkanes may prove to be negatively correlated with the capacity to distinguish between dietary components (see Dove et al., 1999). In such cases, more reliable estimates of diet composition may be obtained if certain alkanes are not used in the calculation (e.g., Lewis et al., 2003), or at least are ‘weighted against’ in minimising the above sum of squares. Theoretically, these would be the alkanes which contribute substantially to the discrepancy between observed and expected concentrations, but little to the actual discrimination of diet components; the issue is how to identify these. This is an issue of current concern and one which requires resolution if plant wax components are to gain wider acceptance as a method for estimating diet composition. Multivariate statistical procedures such as Canonical Variates Analysis (discriminant analysis) or Principal Components Analysis can be used to establish whether plant species can be distinguished and which alkanes are best related to the ability to discriminate between diet components (e.g. Dove et al., 1999; Piasentier et al., 2000). In addition, alkanes with relatively low concentrations in plant materials and in faeces (e.g., even-chain alkanes) are generally measured less accurately than those present at higher concentrations. Hence in choosing which alkanes to use, both their effectiveness in discriminating between plant components and their degree of potential analytical error need to be considered. In species-diverse plant communities, a more usual situation is that there are more plant species available for possible consumption than there are alkanes to discriminate them. Under these circumstances, there are two general approaches to obtaining a diet composition estimate. 1. Decrease the number of ‘effective dietary components’ by grouping species in the diet on some logical basis or, at the extreme, leaving out species altogether. The latter approach may be valid if there are other data which back up the decision to exclude the species (e.g., behavioural or plant-derived data indicating the species is never consumed), but it should be used with caution. The grouping of species is another area in which multivariate statistical pro-

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cedures can be used (e.g., Bugalho et al., 2002, 2004) to indicate that, a priori, it will be difficult to separate some of the available species. This provides an objective justification for grouping those species. Alternatively, species can be grouped on a ‘functional’ basis such as herbage versus browse species (e.g., Bugalho et al., 2002) or on taxonomic grounds (e.g., grasses versus clovers versus Lotus spp.; Kelman et al., 2003). Note that selection by the animals within such groupings may influence the accuracy of the diet composition estimate. In a recent study, Bugalho et al. (2002) found that after grouping the herbage species into a single component, simulated selection within the grouping only had a minor effect on the estimated diet composition. However, this may be a case-specific outcome and if species are grouped for the purposes of estimating diet composition, it would be sensible to carry out some assessment of the impact of possible selection within the groups. 2. Increase the number of ‘discriminators’ used to estimate diet composition; this could be achieved either by combining the use of alkanes (or other plant wax markers) with other methods for estimating diet composition, or by using a number of different groups of plant wax markers. As an example of the former, microhistological and alkane-based methods have been combined to estimate diet composition in sheep which had free access to either Deschampia flexuosa-grassland or Calluna vulgaris (heather)-heathland (Salt et al., 1994). The alkane procedure was used to estimate the proportion of total intake coming from each vegetation association. Microhistological examination of oesophageal extrusa collected from each association was then used to further subdivide the intake into component species. 4.5. Using other plant wax markers to estimate diet composition Of particular interest as additional diet composition markers in plant wax are the alkenes, LCOH and LCFA (Kelman et al., 2003; Ali et al., 2005). These components also have different concentrations in the different plant species and plant parts and can be obtained together with alkanes as part of the same analytical procedure (Ali et al., 2004).

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4.5.1. Alkenes The unsaturated hydrocarbons or alkenes tend to be predominant in the floral parts of plants and have also been found in a number of browse species; they are extracted from plant material and faeces in the same fraction as n-alkanes. The faecal recovery of plant alkenes in sheep is of the order of 25–40% (Dove and Oliv´an, 2005; Elwert et al., 2004; Elwert and Dove, 2005), in contrast with the higher recoveries for alkanes (Fig. 1). A feature of the alkene recoveries is that they show little variation with carbon-chain length, especially from C29 to C33 (Fig. 1). Paradoxically, despite their low faecal recoveries, in a recent study (Dove and Oliv´an, 2005) the alkenes C29 –C33 gave excellent estimates of diet composition, which did not differ from those estimated using alkanes (see Section 5.1.1. below). Moreover, there was no advantage in correcting faecal alkene concentrations for incomplete recovery. This serves to emphasise the point made above that, ultimately, it is the relative recoveries of the markers which are important for estimating diet composition, not the absolute recoveries. In this case, the relative recoveries of alkenes C29 –C33 were so similar that recovery correction did not alter estimated diet composition. 4.5.2. Long-chain alcohols The LCOH are proving to be useful diet composition markers, especially now that appropriate analytical procedures for their routine quantification have been described (Ali et al., 2004). Large between-species differences in LCOH concentrations exist (Table 1) and account for 60–90% of the variance in LCOH concentrations (Bugalho et al., 2004; Ali et al., 2005). In the dicotyledons, concentrations of C30 alcohol are generally much higher than in the monocotyledons, which is likely to be a very useful feature when using alcohol concentrations in diet composition estimates. Whilst primary alcohols predominated in most of the plants analysed, the secondary alcohol, 10nonacosanol, could be a potentially important diet composition marker in studies involving gymnosperms. The faecal recoveries of dietary LCOH have now been determined in two studies and show a progressive increase with chain length similar to that observed with n-alkanes (Fig. 2). In both studies, recoveries have ranged from about 0.6 for C22 -OH to about 0.9 for C30 -OH.

Fig. 2. Influence of carbon-chain length on faecal recovery of LCOH (upper panel) and LCFA (lower panel). Recalculated from the data of Ali et al., 2004 (
) and of H. Dove and E. Charmley () (unpublished data). Vertical bars represent standard errors of mean recoveries.

Bugalho et al. (2004) have recently demonstrated that the discriminatory information provided by the LCOH is additional to that already supplied by the alkanes. As a consequence, plant species were better separated by the combination of alkanes and LCOH than by alkanes alone (Bugalho et al., 2004). The LCOH have now been used successfully to estimate diet composition in both housed (Ali et al., 2005) and grazing sheep (Kelman et al., 2003). 4.5.3. Long-chain fatty acids (LCFA) Procedures for LCFA analysis, as an extension of the procedure used for alkanes and LCOH, have now been published (Ali et al., 2004). The only data available to

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date for faecal recoveries of individual LCFA indicate that, as with other cuticular wax components, recovery increases with increasing chain length (Fig. 2). The widespread incidence of LCFA in plant cuticular wax, the between-species differences in LCFA profile and the faecal recovery data to date suggest that these compounds could also be used as diet composition markers. Ali et al. (2005) reported concentration data for the alkanes, LCOH and LCFA in a range of plant species. The best estimates of diet composition were obtained with a combination of all three marker classes (Ali et al., 2005), indicating that each class of marker provided different discriminatory information.

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Supplement intake has been estimated using markers which are incorporated into and measured in the body water pool of the animal (e.g., tritiated water or lithium salts), or methods involving faecal markers (e.g., chromic oxide, ytterbium salts). We have discussed the features of these methods elsewhere (Mayes and Dove, 2000) and will here restrict our discussion to the possible use of plant wax markers for estimating supplement intake, which is in essence a special case of the estimation of diet composition.

(e.g., Dove and Oliv´an, 1998; Dove et al., 2003), then the intake of supplement can be estimated by treating it as if it were one of the ‘species’ in the diet. First, diet composition is estimated as earlier described; this provides an estimate of the proportion of supplement in the diet. If the animals are also dosed with even-chain alkanes (e.g., C32 ), this allows the estimation of total intake. Diet composition then allows intake to be partitioned into its components, including supplement, thus providing an estimate of supplement intake. A further advantage of this approach is that it can provide an estimate of whole-diet digestibility which accommodates, in individual animals, any interactions of supplement and herbage during digestion. If the supplement is itself based on herbage (e.g. hay, silage or pelletted herbage), then the natural alkanes of the supplement can be employed without further manipulation. However, in some cases (e.g. legume grains, coarse grains, oilseed meals), the levels of alkane may be insufficient to permit the estimation of diet composition. Under these circumstances, supplements can be labelled with an external source of hydrocarbons, such as beeswax (Dove and Oliv´an, 1998; Dove et al., 2002b). Validation studies (Dove and Oliv´an, 1998; Fig. 3) have shown that this provides accurate estimates of supplement intake. Moreover, as described above, supplement intake estimates were equally good when based on the unsaturated hydrocarbons (alkenes; Dove and Oliv´an, 2005). We must re-emphasise that if the diet composition estimate is in error, so will be the estimate of total intake. This means that care must be taken in estimating the diet composition itself. Note also that to obtain an estimate of total intake and thus of supplement intake, it is necessary to dose the animals with a source of even-chain alkanes. However, recent studies indicate that estimates of forage intake could be obtained without separately dosing the animals.

5.1.1. Using plant wax alkanes to estimate supplement intake Individual even-chain alkanes have been incorporated into supplements and used as supplement intake markers (e.g., Dove and Oliv´an, 1998; Hameleers and Mayes, 1998) but in general, there has been much greater success in using alkane profiles to estimate supplement intake. If the supplement has its own alkane profile or can be labelled with a distinctive profile

5.1.2. Using supplement intake to estimate forage intake In the above examples, plant wax marker profiles were used to estimate diet composition, which then allowed the calculation of total intake and thus supplement intake in dosed animals. However, there are a number of situations in which the intake of supplement by individual animals either is known or could be known with relatively little trouble, e.g., animals

5. Estimation of supplement intake Most sheep and goats rely heavily on sown or natural herbage or browse as their source of nutrients, but there are times of the year when the supply or quality of herbage is inadequate for the production target and animals are offered supplements. Interpretation of animal responses and the interaction between herbage and supplement intakes would be much easier if individual intakes of supplement were known. 5.1. Approaches to estimating supplement intake

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Intake of the forage component of the diet (If ) is then given by (It − Is ). 2. This method for two-component diets (forage + supplement) can be restated in more general terms to allow the estimation of the intake of all the forage components of the diet, in situations where the animals are consuming known amounts of supplement and unknown amounts of a number of forage species. By rearrangement of the above terms, it can be shown that If can be estimated directly as   pf (8) If = Is × ps where pf is the proportion of forage in the diet. The advantage of the approach embodied in Eq. Fig. 3. Relationship between known supplement intakes and those estimated using beeswax as an alkane marker to estimate supplement proportion in the diet, and natural and dosed alkanes to estimate total intake (adapted from Dove and Oliv´an, 1998). Supplement intakes estimated using alkenes (unsaturated hydrocarbons) were related to known intakes by the expression: Y = 1.057X − 0.010 (r2 = 0.995) Alkane-based and alkene-based estimates were related by the expression: Y = 1.010X + 0.000 (r2 = 0.994). Neither of these expressions, nor that in the above figure, differed significantly from Y = X.

receiving known amounts of supplement while in the dairy parlour. If these supplements either contain or can be labelled with alkanes, the proportion of supplement in the diet can be estimated as above. However, because actual supplement intake is also known, total and thus herbage intake can also be computed. This approach provides a means of estimating the intake of pasture or its component species, without having to dose animals with alkanes, which in turn may avoid undue stress or perturbations of normal grazing behaviour. The procedure for estimating intakes in this way is as follows. 1. Estimate diet composition as described above, using the alkane profiles of diet components and of faeces (recovery-corrected). In a two-component diet (forage, supplement), if the known supplement intake is Is , and the proportion of supplement in the diet is ps , then the total intake It is given by It =

Is ps

(7)

Fig. 4. Comparison of known forage intakes and the intake of (a) a single forage (Dove et al., 2002b) or (b) multiple forages (Dove et al., 2003), calculated using the estimated proportions of forage and supplement in the diet, together with known supplement intake. In both cases, the fitted regressions did not differ from the line of equality (solid lines).

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(8) is that it should be equally applicable to multicomponent diets (supplement, forage species 1, forage species 2, . . ., forage species n). A further and major advantage is that the intake of the various forage species (and/or plant parts) is obtained without the separate dosing of the animals. In effect, all of the alkanes in the supplement are used as the dose. The use of supplement intake to estimate the intake of the forage components of the diet was validated for two-component diets by Dove et al. (2002b; Fig. 4a) and by Elwert and Dove (2005), and for multi-component diets by Dove et al. (2003; Fig. 4b).

6. Conclusions In sheep and in a single study with goats, the alkane procedure for estimating forage intake is now well validated. Under field conditions, it has been compared with previous techniques such as the Cr-in vitro procedure. While no simple relationship between these approaches should be expected, we conclude that the alkane-based estimates are likely to be more accurate, because they accommodate differences in forage digestibility between individuals, and those arising from interactions between supplement and forage. A further (and major) advantage of the use of plant wax markers is that they also allow an estimate of diet composition and thus the partitioning of total intake into its component plant species. When used alone, alkanes can discriminate between fewer species than is often encountered by sheep or goats utilising complex plant communities. However, we conclude that the LCOH and LCFA of plant wax can now also be used as diet composition markers. This, plus the use of multivariate statistical procedures to guide the grouping of species within the possible diet, should extend substantially the number of species which can be discriminated in the diet. In addition, other compounds in plant wax, as yet unevaluated as markers, could further extend diet composition estimates. Supplement intake can also be estimated using wax markers, and we conclude that there is considerable scope for exploiting known supplement intakes as a means of estimating the intake of other diet components, thus avoiding the need to separately dose the test animals with synthetic alkanes.

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