R.W. Mayes

Measurement of dietary nutrient intake in free-ranging mammalian herbivores

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-fistulated (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 briefly discuss methods based on grazing behaviour measurements or on H2O 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 unidentifiable. 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 composition measurements to the measurement of nutrient transactions within the gut, particularly in relation to the supply of absorbable nutrients.

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.

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 effects of season and diet composition on the radiocaesium intake by sheep grazing on heather moordland

In north-east Scotland on a mixed heather and grassland pasture grazed by sheep, 2 × 2 m areas of Calluna vulgaris heath and Deschampsia flexuosa grassland were artificially contaminated with 134 Cs by soil injection. Estimates of the 134 Cs intake by sheep were made on the basis of measurements of dry matter intake, diet composition and 134 Cs concentrations in diet components, assuming that the whole pasture had been contaminated. Dry matter intake by sheep was measured using a natural n-alkane of plant waxes and an orally administered n-alkane as markers. Diet composition was determined by a combination of botanical analysis of samples of ingested material and relationships between n-alkane patterns in faeces and ingested material (...)

Plant wax alkanes and alcohols as herbivore diet composition markers

Abstract The n-alkanes in plant cuticular wax have been used as markers for estimating the species composition of herbivore diets, but the long-chain fatty alcohols (LCOH) of plant wax may also be useful. The objective of this research was to assess if LCOH contributed extra information to differentiate plant species, compared with n-alkanes only. We used 3 data sets consisting of n-alkane and LCOH concentrations of plant species occurring in pastures of New South Wales, Australia. We used Principal Component Analysis (PCA) to summarise the data for n-alkane and LCOH concentrations obtained for the species in these data sets. The first 3 principal components explained 86 to 93% and 75 to 99% of the variance in n-alkane and LCOH concentrations, respectively. Orthogonal Procrustes Rotation (OPR) was then used to compare the results of PCA conducted with n-alkane and LCOH data, with a view to establishing whether LCOH provided discriminatory information in addition to that provided by the n-alkanes. Results of OPR indicated that this was so for all 3 data sets, and suggested that the LCOH would be useful additional markers for discriminating between plant species. We tested this by using Discriminant Analysis and cross-validation procedures in 2 data sets to distinguish between defined species groups of C3 grasses, C4 grasses, clovers and Lotus spp. The discrimination between these categories and the proportion of plant species correctly classified into the defined categories was better when using n-alkanes and LCOH together, compared with alkanes alone. Our results indicate that LCOH provided additional information that could be used for distinguishing plant species as part of estimating the species composition of herbivore diets.

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

Previous 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 seasons 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 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.

The flexibility of an intermediate feeder: dietary selection by mountain hares measured using faecal n-alkanes

Herbivores with an intermediate feeding strategy either vary their diet between a grazing (bulk roughage feeders) or browsing (concentrate selectors) strategy on a seasonal basis or select a mixed diet at any one time. The underlying ecological causes of the seasonal dietary shift in a small non-ruminant intermediate feeder – the mountain or arctic hare (Lepus timidus L.) were determined. Diet composition and selection relative to availability were investigated for 41 individual free-ranging mountain hares (of which 18 female hares were radio-collared) occupying an upland mosaic landscape in north-east Scotland. Diet composition was determined using faecal n-alkane analysis. Radio-collared hares were designated as pasture, woodland or moorland hares according to the habitat that predominated their home-range. In common with previous studies, mountain hares switched from a browse-dominated diet during winter to a Gramineae-dominated diet in summer, although it was only significant for reproductively active females during the peak breeding season. Diet composition remained consistent regardless of habitat occupied. However, the diet of radio-tracked hares differed significantly from the biomass available in the individual home-ranges; Gramineae were preferentially selected over browse species throughout the year. During winter and in particular during the early breeding season, intermediate feeders, such as mountain hares, ate browse material when the availability of higher quality was restricted. The ability to browse or graze represents a flexible foraging strategy permitting survival and production through periods of changing or unpredictable forage quality and availability.

Novel approaches to the estimation of intake and bioavailability of radiocaesium in ruminants grazing forested areas

It is difficult to measure transfer of radiocaesium to the tissues of forest ruminants because they can potentially ingest a wide range of plant types. Measurements on undomesticated forest ruminants incur further difficulties. Existing techniques of estimating radiocaesium intake are imprecise when applied to forest systems. New approaches to measure this parameter are discussed. Two methods of intake estimation are described and evaluated. In the first method, radiocaesium intake is estimated from the radiocaesium activity concentrations of plants, combined with estimates of dry-matter (DM) intake and plant species composition of the diet, using plant and orally-dosed hydrocarbons (n-alkanes) as markers. The second approach estimates the total radiocaesium intake of an animal from the rate of excretion of radiocaesium in the faeces and an assumed value for the apparent absorption coefficient. Estimates of radiocaesium intake, using these approaches, in lactating goats and adult sheep were used to calculate transfer coefficients for milk and muscle; these compared favourably with transfer coefficients previously obtained under controlled experimental conditions. Potential variations in bioavailability of dietary radiocaesium sources to forest ruminants have rarely been considered. Approaches that can be used to describe bioavailability, including the true absorption coefficient and in vitro extractability, are outlined.

Phthalate and alkyl phenol concentrations in soil following applications of inorganic fertiliser or sewage sludge to pasture and potential rates of ingestion by grazing ruminants

Soil concentrations of dioctyl phthalate (DOP) and the alkyl phenols, octyl phenol (OP) and nonyl phenol (NP), after repeated surface applications of sewage sludge to pastures, were investigated. Liquid sludge was applied at a rate of 2.25 tonnes dry matter (DM) per hectare to each of three treated (T) plots on three occasions during the summer and two occasions in the early spring over a period of 2.5 years. Control (C) plots were treated with inorganic fertiliser containing amounts of nitrogen equivalent to those applied to the treated plots. At between 69 and 81 days after the application of sludge, 15 separate soil samples were collected from one half of each of the plots (Experiment 1). Concentrations (microg g(-1)) of DOP were higher (P < 0.001) than those of NP, while those of OP were generally below detectable levels. Mean soil concentrations of DOP were not significantly different in T and C plots [0.233 vs. 0.155 microg g(-1); standard error of the difference (SED) = 0.046; not significant (NS)], partly because there was already a relatively large amount of DOP present. NP concentrations were, however, significantly higher in T than in C plots (0.021 vs. 0.013 microg g(-1) SED = 0.002; P < 0.05). There was no consistent change over time in the mean soil concentrations of these compounds when sampled at intervals of 3-6 months. Concentrations in soil samples collected at monthly intervals following sludge application indicated that the variation in concentrations of these endocrine-disrupting compounds (EDC) was unrelated to time since sludge application. Rates of soil ingestion, expressed as the percentage of DM intake represented by soil, were higher during the winter than the summer (5.40 vs. 1.17; SED = 0.360; P < 0.001) and estimated daily intakes of DOP and NP were up to 150 microg and 8 microg, respectively. It is concluded that the application of sewage sludge to pasture does not increase soil concentrations of phthalate (as DOP) or alkyl phenols. Thus, the risk of increased exposure to these EDC as a result of sludge application is small. However, the small effect of sludge application on soil concentrations may be largely a reflection of the relatively high concentrations of DOP already present in the soil, which may be biologically significant.

The possible use of n-alkanes, long-chain fatty alcohols and long-chain fatty acids as markers in studies of the botanical composition of the diet of free-ranging herbivores

Few methods exist for estimating quantitatively the diet composition of free-ranging herbivores. The current study examined whether long-chain fatty alcohols (alcohols) or long-chain fatty acids (acids) could be used along with n-alkanes to allow reliable diet composition estimates to be made in herbivores consuming complex diets. Twelve Scottish Blackface wether sheep housed in metabolism crates were fed four different mixtures of three plant species (three animals per mixture) for a seven-period experiment. Concentrations of cuticular wax n-alkanes, alcohols and acids were estimated in samples of individual plant species, and the faeces from animals that consumed mixtures of these species. These concentrations were then used to calculate the dietary proportions of each species by a least-squares optimization procedure. To explore the differences between the estimation methods (individual markers and their combinations), the mean squares of errors (EMS) between the actual and predicted proportions of plant species were calculated. In three out of the four mixtures, alcohols had the lowest discrepancies (lowest EMS values), followed by n-alkanes and then acids. Acids yielded the lowest discrepancy in one mixture and the highest in the others. It is concluded that, for this particular set of mixtures, alcohols had great potential to estimate composition of complex diets. However, the estimation using acids was less good and n-alkanes were of intermediate potential. Estimation from the combination of the three marker classes was always better than using the poorest individual marker.

The effect of treating pastures with bentonite on the transfer of 137Cs from grazed herbage to sheep

Abstract The persistence of high levels of 134 Cs + 137 Cs in vegetation of certain upland areas of the United Kingdom has prompted investigations into possible methods of reducing levels in sheep tissues. The introduction of the clay mineral, bentonite, into the diet of ruminants has been shown to reduce the uptake of caesium from the gut. The effects of treating pasture with bentonite at two rates (single or repeated application of 80 g m −2 ) on the intake and transfer of 137 Cs to sheep tissues was investigated. Whilst there were reductions in the radiocaesium activity concentrations of tissues of sheep grazing pasture treated at both rates, transfer coefficients were only affected for sheep on the repeated treatment. However, ewes grazing the repeatedly treated pasture experienced a loss in body weight (18%) associated with a decrease in herbage intake (39%). Considerable variation in the herbage intake by individual ewes and lambs, both between and within treatments, showed the need for direct intake measurements in order to determine relevant transfer coefficients in field experiments. Transfer coefficients for 137 Cs of 0.33 d kg −1 and 1·61 d kg −1 were obtained for muscle of control ewes and lambs respectively.