SUMMARYThe recovery in the faeces of the n-alkanes of herbage (odd-chain, C27–C35) and of dosed artificial alkanes (even-chain, C28 and C32) was studied in twelve 4-month-old castrated male lambs. The lambs received three levels of cut, fresh perennial ryegrass or a mixed diet of perennial ryegrass (0·70) and a barley-based concentrate (0·30) (500–900 g D.M./day). C28 and C32 n-alkanes (130 mg each), absorbed onto shredded paper, were given once daily for 17 days to test whether the recoveries of herbage and dosed alkanes were similar to enable their use as markers for determining the herbage intake of grazing sheep. Stearic and palmitic acids (130 mg each) were given with the dosed alkanes to half of the animals with the objective of facilitating emulsification of the dosed alkanes within the digestive tract.With the exception of C27 n-alkane, the faecal recoveries of all alkanes were unaffected by diet, feeding level or emulsifying agent. Faecal recovery of odd- chain herbage n-alkanes increased with increasing C-chain length. The recovery of the dosed C28 n-alkane was slightly greater than the recoveries of both C27, and C29 n-alkanes of herbage. The recoveries of the dosed C32 n-alkane and the herbage C33-alkane were the same.The mean herbage intake estimated using C33 and C32 n-alkanes was identical to the actual herbage intake. Other alkane pairs gave slight underestimates of herbage intake ranging from 3·5% for the C28–C29 pair to 7·6% for the C27–C28 pair. No cyclical pattern of n-alkane excretion throughout the day was observed. Examination of daily variations in faecal alkane concentrations indicated that the start of alkane dosing should precede the sampling of faeces by at least 6 days.These results suggest that accurate estimation of herbage intake in grazing sheep is possible from the simultaneous use of dosed C32 and herbage C33 n-alkanes as markers.The method may be particularly useful in enabling unbiased estimates of herbage intake to be made in animals receiving supplementary feed.

This review discusses the potential use of plant wax components, especially n-alkanes, as markers for estimating herbage intake, estimating the botanical composition of consumed herbage and studying digesta kinetics. Previous approaches to making these measurements are discussed briefly. Attention is drawn to the fact that current methods for estimating intake do not adequately allow for differences between individual animals. It is also suggested that the markers currently used to estimate botanical composition or study digesta kinetics are inadequate. The nature of the chemical constituents of plant waxes is briefly discussed and the concept of using alkanes to estimate intake is introduced. Particular emphasis is given to the fact that although the recovery of alkanes in faeces is not complete, intake can still be estimated using a pair of alkanes (one natural, one dosed) provided these have similar faecal recoveries. The accuracy of estimation of intake is discussed in terms of: obtaining a representative sample of herbage; alkane dosing and faecal sampling procedures; validity of the assumption of similar recoveries for the natural and dosed alkanes; sample preparation and analysis. Published comparisons of estimated and actual intakes are presented, with the conclusion that satisfactory results are obtained if intake is estimated using natural C33 alkane and dosed C32 alkane. The use of the different patterns of alkanes in herbage species, as a means of estimating botanical composition, is then discussed. Results are presented showing this can be done successfully with herbage mixtures or oesophageal extrusa. Procedures are then described for making the corrections for incomplete faecal alkane recovery, necessary to estimate the botanical composition of the herbage consumed by the free-grazing animal. This allows the quantification of the intake of individual plant species by individual animals, and it is suggested that this can be achieved without the need for oesophageally-fistulated (OF) animals. Differences in alkane levels between plant parts within a species are then discussed. It is suggested that these can lead to error in the estimation of intake, if OF animals should consume plant parts different from those consumed by the test animals. However, it is also suggested that differences in alkane levels between plant parts can be used to quantify the intake of these parts, in a manner analogous to the estimation of the intake of individual plant species. The usefulness of alkanes in studies of digesta kinetics is then discussed, principally in relation to the natural alkanes, which remain intimately associated with plant particles in the gut. It is suggested that natural alkanes could prove excellent markers for studies of particle breakdown and digesta flow. The preparation of natural 14C-labelled alkane, for use as a pulse dose in mean retention time studies, is also discussed.

The nutrient status of the herbivore depends on the nutritive value of the plants available, the botanical composition of the consumed diet and the intake of the animal. It has always been difficult to quantify these last two. At present, intake is usually calculated from separate estimates of fecal output and diet digestibility. In this review we discuss the errors inherent in this approach, especially those associated with the determination and application of digestibility in vitro. We then critically evaluate a new approach to the estimation of intake, based on the use of plant cuticular wax alkanes as markers. Plant alkanes are predominantly odd-chain and substantially indigestible. They can be used, in combination with orally dosed even-chain alkanes, to obtain an intake estimate which is essentially independent of marker recovery in feces and which is more truly "individual" because it accommodates the level of digestibility occurring in individual animals. We present published data which indicate that the method is accurate and can be extended to measure diet composition as well. Previous approaches to estimating diet composition have been based on the laborious microscopic examination of esophageal extrusa, stomach contents or feces. However, most plant species have a characteristic pattern of alkane concentrations in their cuticular wax. This permits the estimation of diet composition from the pattern of alkanes in the feces and in the plants available. We present data to show that this approach can provide accurate estimates of diet composition in terms of either plant species or plant parts. A major advantage of the approach is that, if the animals are also dosed with even-chain alkanes, estimates of total intake and diet composition can be obtained simultaneously. The method is equally applicable to domestic and wild herbivores and to animals receiving supplementary feeds. In future work, the method will be extended to the simultaneous estimation of plant species and plant parts in the diet, and to the use of other wax components as markers.

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Plant-wax markers can be used for estimating forage intake, diet composition and supplement intake in grazing livestock, wild ruminants and other mammals. We describe protocols for using the saturated hydrocarbons (alkanes) of plant wax as markers for estimating fecal output, intake and digestibility. Procedures for investigating digestion kinetics are also discussed. Alkanes can also be used to estimate diet composition and the procedures required to do this are also described, including the special case where supplementary feed is treated as a component of the diet composition estimate. The long-chain alcohols (LCOHs) and very long-chain fatty acids (VLCFAs) of plant wax show particular promise for discriminating a greater number of species in the diet. The use of all these plant-wax markers in nutrition studies depends on having quantitative, repeatable and mutually compatible assay procedures for alkanes, LCOHs and VLCFAs; we present protocols for these assays in detail. Analysis of a single sample of feces or plant material for all these plant-wax markers can be completed within 2 days; however, it is possible to process up to 50 samples (analyzed in duplicate) per week.

The nutrient intakes of mammalian herbivores depend on the amount and the nutrient content of the plant species and plant parts which they eat. We review the merits of oesophageal-®stulated (OF) animals, microhistological procedures, stable C-isotope discrimination and plant cuticular-wax markers as methods for estimating diet composition and intake in both ruminant and non-ruminant herbivores. We also brie¯y discuss methods based on grazing behaviour measurements or on H 2 O or Na turnover, and methods for estimating supplement or soil intake. Estimates of intake in ruminants are often based on separate measurements of faecal output and herbage digestibility. We review this approach and emphasize that, under some circumstances, the applicability of in vitro digestibility estimates based on OF extrusa is questionable. We discuss how plant-wax marker patterns can be used to check whether OF and test animals are consuming similar diets, but also emphasize that a major advantage of the use of plant-wax markers is that this approach may obviate altogether the need for OF animals. Estimates of total herbage intake can be partitioned into the intakes coming from different plant species and=or parts, provided diet composition can be measured. Diet composition estimates based on C-isotope discrimination have the major disadvantage that they cannot be taken to species level. By contrast, microhistological methods can identify many plant species in extrusa, digesta or faeces, but often a large proportion of plant fragments remains unidenti®able. Plant-wax hydrocarbons show great promise as markers for estimating diet composition and intake. However, we suggest that to be applicable in complex plant communities there is a need with this method either to recruit a wider range of wax markers (e.g. alcohols, sterols, fatty acids) or to use it in combination with other methods. We suggest that, in turn, this generates an urgent need for research on statistical aspects of the combined use of markers or methods, in relation to the error structures of the data or methods being combined and the standard errors of the resultant estimates of diet composition and intake. We conclude by discussing the extension of intake and=or diet Abbreviation: OM, organic matter.

SU MMARYPrevious investigations have shown that the long-chain fatty alcohols and long-chain fatty acids of plant waxes have potential as diet composition markers. This study was conducted to measure faecal recoveries of long-chain fatty alcohols (C 20 -C 30 ) and long-chain fatty acids (C 20 -C 32 ) in sheep fed mixed diets. Methodology for quantitative analysis of these compounds in feed and faeces is also presented. The method was an extension of the original n-alkane method of Mayes et al. (1986) in which separate hydrocarbon (n-alkanes, n-alkenes and branched-chain alkanes), alcohol (free+ esterified) and acid (free+esterified) fractions could be obtained from a single sample. A fraction containing alcohols and sterols was eluted from the silica gel column after removal of the hydrocarbons. Sterols were removed from alcohols using aminopropyl solid-phase extraction columns. Alcohols were converted to their trimethylsilyl (TMS) ethers and run on a gas chromatograph (GC). Acids were extracted from the aqueous phase of saponification products after removal of hydrocarbons, alcohols and sterols, purified through silica gel columns and were converted into their methyl esters (FAMES) prior to analysis on a GC. Tests were carried out to evaluate the reproducibility of the results obtained from the analytical method developed for quantifying alcohols and acids. Twelve sheep, in metabolism crates, were offered (0 . 8 kg DM/animal/day) four different mixtures of hill grass (Agrostis capillaris), birch (Betula pendula) leaves and current season's growth of heather (Calluna vulgaris) and bilberry (Vaccinium myrtillus) for 17 days. Total daily faeces and feed refusals collections were carried out over the last 7 days. Faeces collections were bulked for each animal. Representative samples of feed, refusals and faeces were analysed for alcohols and acids using the described method. Faecal recoveries of alcohols and acids were calculated from the ratio of output and input of each marker. The results showed high, though incomplete, faecal recoveries for both alcohols and acids. Alcohols had consistently higher faecal recoveries compared with acids. Mean (¡S.E.) faecal recovery values for alcohols C 20 , C 22 , C 24 , C 26 , C 28 and C 30 were 0 . 58¡0 . 04, 0 . 67¡0 . 01, 0 . 72¡0 . 008, 0 . 80¡ 0 . 007, 0 . 94¡0 . 005 and 1 . 01¡0 . 02, respectively, whereas those of acids C 20 , C 22 , C 24 , C 26 , C 28 , C 30 and C 32 were 0 . 47¡0 . 02, 0 . 57¡0 . 02, 0 . 61¡0 . 02, 0 . 77¡0 . 017, 0 . 84¡0 . 01, 0 . 79¡0 . 015 and 0 . 84¡0 . 013, respectively. Increasing chain-length had a significant effect (P<0 . 05) on the recoveries of both alcohols and acids (R 2 =0 . 808, 0 . 741, respectively). Different dietary plant mixtures had no effect (P>0 . 05) on the recoveries of alcohols and acids in faeces.

In 1993 and 1994, 40 cows in early lactation in early spring were assigned randomly to four feeding treatments. One group of cows was kept indoors with access to grass silage ad libitum, plus 6 kg of concentrate daily. The other three groups had access to grass pasture (5–6 h per day in 1993 and 11–12 h per day in 1994) plus grass silage similar to that fed to the previous group while indoors plus 6, 4 or 2 kg of concentrate daily. The average daily allocations of herbage (> 3·5 cm) were 8·5 and 14·0 kg DM cow−1 day−1 in 1993 and 1994 respectively. The treatments were applied for 8 weeks (26 February to 23 April) in 1993, and 7 weeks (11 March to 29 April) in 1994. Cows with access to pasture had lower (P < 0·001) silage dry‐matter (DM) intakes and higher (P < 0·001) total forage DM intakes in both years than those kept indoors. This resulted in significantly higher yields of milk, fat, protein and lactose. Similarly, milk protein concentration was higher (P < 0·05 in 1993; P < 0·001 in 1994). There was a significant linear increase in total DM intake in both years with increased concentrate supplementation. In 1993, there was a linear increase in milk (P < 0·01), fat (P < 0·01), protein (P < 0·001) and lactose (P < 0·01) yields with increased concentrate supplementation. In 1994, only milk protein yield (P < 0·05) was increased. Concentrate supplementation had no effect on milk composition or liveweight change. Cows with access to grazed grass had higher liveweight gains (P < 0·05) than those kept indoors in both years. In 1993, increasing the energy intake increased the processing qualities of the milk produced. The results showed that access to grass pasture resulted in higher milk production, in reduced silage requirement and in reduced level of concentrate supplementation required for a given level of milk production with spring‐calving cows in early lactation compared with those kept indoors.