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

Abstract

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 (C₂₀–C₃₀) and long-chain fatty acids (C₂₀–C₃₂) 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 C₂₀, C₂₂, C₂₄, C₂₆, C₂₈ and C₃₀ 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 C₂₀, C₂₂, C₂₄, C₂₆, C₂₈, C₃₀and C₃₂ 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 (R²=0·808, 0·741, respectively). Different dietary plant mixtures had no effect (P>0·05) on the recoveries of alcohols and acids in faeces.