Cuticular wax alkanes and alcohols used as markers to estimate diet composition of sheep (Ovis aries)

The chemical components in plant cuticular wax can be used as markers to estimate the species composition of the diet of grazing animals. In this experiment, sheep (Ovis aries) grazed swards consisting of two sown species of Lotus (L. corniculatus and L. pedunculatus), two sown grasses (Phalaris aquatica and Austrodanthonia richardsonii) and volunteer annual grasses and legumes (dominated by Vulpia myuros and Trifolium spp.). The botanical composition of the sward was estimated before and after grazing over three days by four sheep. During the grazing period, samples of whole plants of the species present, plus rectal faecal samples from the sheep, were collected, freeze-dried and ground prior to the extraction of cuticular wax n-alkanes and long-chain alcohols. Purified alkanes and alcohols in samples of faeces and plants were quantified by gas chromotography. The species composition of the sheep diets was estimated by relating faecal alkane and alcohol contents to those of the plant species on offer, using the 'EatWhat' software package. Principal Components Analysis explained 75% of the variance in the pattern of alkane concentrations between the species on the first two axes. PCA using alcohols accounted for 68% of the variance on the first two axes and separated the Lotus species from the grasses and clovers. Comparison of the two PCA, using Orthogonal Procrustes Rotation, indicated that the long-chain alcohols contributed extra information over and above that provided by the alkanes. Discriminant analysis of alkane and alcohol concentrations enabled a clear separation of Lotus, clover and grass species groups. Using alkanes as markers, the diet consumed by sheep contained 18+/-1.1% Lotus, whilst using both alkanes and alcohols, the estimated Lotus content of the diet (26+/-1.2%) was closer to the Lotus content of the sward (31%). Diet composition estimates indicated that the Lotus component consisted mostly (>90%) of L. corniculatus. This apparent selection against L. pedunculatus may relate to the much higher condensed tannin content in that species.