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

Abstract

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.

Application of long-chain alcohols as faecal markers to estimate diet composition of horses and cattle fed with herbaceous and woody species

“…In general, herbaceous species are known to possess very low total alkane concentrations (Dove and Mayes, 2006;Oliván et al, 2007b). In comparison with LCFA, LCOH showed lower total concentrations as observed by Ferreira et al (2015) with similar plant species.…”

“…After the alkane analysis, all samples were stored in plastic containers under vacuum. The LCFA and LCOH contents of faeces and dietary plant species were analysed in duplicate according to the methods of Dove and Mayes (2006). Analysis of LCOH (C 20 -C 30 ) was carried out by gas chromatography, using a Carlo Erba Mega Series 5160 (Carlo-Erba, Milan, Italy) fitted with flame ionization detector.…”

“…As reviewed by Dove and Mayes (2006), this is a non-invasive approach offering several potential advantages over traditional methods. Nevertheless, the limited number of plant species that can be discriminated and the low alkane concentrations in some plant species have been pointed out as the main limitations for application of these markers.…”

“…Nevertheless, the limited number of plant species that can be discriminated and the low alkane concentrations in some plant species have been pointed out as the main limitations for application of these markers. In fact, some plant species that can be found on heathland vegetation communities with associated improved pastures (Lolium perenne, Trifolium repens, Pseudarrhenatherum longifolium, Agrostis capillaris and Ulex gallii ) present very low alkane concentrations (Ferreira et al, 2009a and, making them more prone to measurement errors (Dove and Mayes, 2006). Thus, their application may result in less accurate estimations of both diet selection and feed intake (Ferreira et al, 2007).…”

In vitro studies of the metabolism of [14C]-n-alkanes using ruminal fluid of sheep as substrate

“…Extraction of n-alkanes from residues of reactions was performed in the same Pyrex tubes used for in vitro incubations, using n-docosane (C 22 ) and n-tetratriacontane (C 34 ) as internal standards (0.1 g of a solution containing 0.2 mg of each hydrocarbon per gram. For details, see Mayes et al (1986) as modified by Dove and Mayes (2006)). After evaporation of the filtrate, 2 ml of a solution of n-heptatriacontane (C 37 ) in n-heptane (0.02 mg/g) was added to the bottles as an additional standard in order to quantify any losses of C 34 occurring during the extraction process.…”