Technical note: Analytical refinements of the methane indicator archaeol in bovine feces, rumen fluid, and feedstuffs

摘要

Archaeol (1,2-di-O-phytanyl-sn-glycerol) is a cell membrane lipid component of methanogens that has the potential to be used as a biomarker for methane production in ruminants. However, its analysis via gas chromatography-mass spectrometry (GC-MS) is challenging because of its molecular size and structure. In this study, 2 different sample preparation methods were tested, Soxhlet and sonication-aided extraction, and the methods were compared for extraction efficiency using the internal standard (IS; 1,2-di-o-hexadecyl-rac-glycer-ol). The extraction efficiency of the Soxhlet method for fecal archaeol was twice that of sonication. With the use of a high-temperature GC column, the retention times of IS and archaeol were 17.6 and 19.4 min, respectively, with a total run time of only 25 min. The molecule ions m/z 611.4 (IS) and m/z 725.8 (archaeol), or alternatively the fragment ion of the glycerol moiety m/z 130.0, were used for identification and quantification via GC-MS in positive chemical ionization mode. The intra-assay coefficients of variation for fecal archaeol measurements were 1.3% (m/z 725.8) and 2.1% (m/z 130.0) (n = 3), respectively. Fecal archaeol quantifications did not differ between the use of the molecule or glycerol moiety ions (paired t-test, n = 156). Archaeol concentrations tended to be 3.3% greater in samples stored at -20℃ before drying compared with samples that were immediately dried after collection (paired t-test, n = 5). The detection limit of archaeol was 0.5 μg/g of fecal dry matter (DM); no archaeol could be detected in feed samples. In different fractions of rumen fluid, archaeol levels ranged from 1.9 to 24.0 μg/g of DM. In 10 cows fed the same grass and corn silage/ hay-based ration, diurnal variations of fecal archaeol levels (5 time points over 2 d) were cow dependent and ranged from 26.2 to 77.2 μg/g of DM (mean 48.4 μg/g of DM). Thus, within-animal variation in cows on the same diet was between 4 and 27%. We suggest that this finding is related to the amount and time of the latest feed intake event before the fecal sampling. Feeding pattern can determine the passage rate of digesta through the alimentary tract and thus the duration of contact time of