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

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.

Using the alkanes and long-chain alcohols of plant cuticular wax to estimate diet composition and the intakes of mixed forages in sheep consuming a known amount of alkane-labelled supplement

“…By contrast, LCOH concentrations in the diet components were much higher than their alkane concentrations. This has been reported previously (Ali et al, 2004;Bugalho et al, 2004;Ali et al, 2005). In general, the LCOH concentrations we report confirm earlier reports (Bugalho et al, 2004;Ali et al, 2005;Fraser et al, 2006) that the predominant LCOH of grass species are C26OH and C28OH.…”

“…The DM content of feed offered, feed refused and faeces was determined by forced-air oven drying at 708C for 48 h. Organic matter (OM) was determined as the weight lost upon ashing feeds and faeces at 5508C in a muffle furnace for 24 h. Alkanes and LCOH were extracted from samples using a direct saponification procedure followed by column chromatography (Ali et al, 2004;Dove and Mayes, 2006) and quantified by gas chromatography using a modification (Salt et al, 1992) of the method of Mayes et al (1986) for alkanes and for LCOH, the method of Ali et al (2004) except that LCOH were not derivatised prior to chromatography. Tetracosane (C24 alkane) and tetratriacontane (C34 alkane) were used as internal standards for alkanes and heptacosanol (C27OH) as the internal standard for LCOH.…”

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

“…In this study, the most accurate estimates of diet composition for both species were obtained when using the mean faecal concentration for the total of animals within species. These results are consistent with those reported by previous studies for horses and cattle with alkanes (Ferreira et al, 2009a) and LCFA markers (Ferreira et al, 2010), and for small ruminants with LCOH markers (Ali et al, 2004;Dove and Charmley, 2008;Ferreira et al, 2012 and2015) and highlight the need of using accurate estimates of markers' FR to improve the accuracy of diet composition estimates in both horses and cattle.…”

“…For cattle, a positive association between CCL and LCOH FR was found. A similar trend was observed for horses and cattle when using LCFA (Ferreira et al, 2010) and alkanes (Ferreira et al, 2007 and2009a), and also in other ruminant species when studying LCOH (Ali et al, 2004;Dove and Charmley, 2008), LCFA (Ali et al, 2004;Ferreira et al, 2009b and and alkanes (Dove and Mayes, 2005;Ferreira et al, 2005 and2009a;Charmley and Dove, 2007). These data indicated that markers behave differently in the gastro-intestinal tract of ruminant and nonruminant species, and suggest that the disappearance of those with the shorter carbon-chain in the gut of ruminants occurs to a greater extent than in non-ruminants.…”

“…Thus, their application may result in less accurate estimations of both diet selection and feed intake (Ferreira et al, 2007). To overcome these limitations, other epicuticular wax components such as long-chain fatty acids (LCFA) (Ali et al, 2005;Ferreira et al, 2009b;Lin et al, 2009), and long-chain fatty alcohols (LCOH) (Ali et al, 2004;Ferreira et al, 2012 and2015) have been suggested as additional markers to estimate diet composition of small ruminants. In fact, their utilization may increase the accuracy of estimates by providing additional discriminatory information to that supplied by alkanes.…”