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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.

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Figure 1: Fecal excretion curves of herbage alkane.
Figure 2: Gas chromatograph trace.

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References

  1. Mayes, R.W. & Dove, H. Measurement of dietary nutrient intake in free-ranging mammalian herbivores. Nutr. Res. Rev. 13, 107–138 (2000).

    CAS  Google Scholar 

  2. Kotb, A.R. & Luckey, T.D. Markers in Nutrition. Nutr. Abs. Rev. 42, 813–845 (1972).

    CAS  Google Scholar 

  3. Dove, H., Mayes, R.W. & Freer, M. Effects of species, plant part, and plant age on the n-alkane concentrations in the cuticular wax of pasture plants. Aust. J. Agric. Res. 47, 1333–1347 (1996).

    CAS  Google Scholar 

  4. Dawson, L.A., Mayes, R.W., Elston, D.A. & Smart, T.S. Root hydrocarbons as potential markers for determining species composition. Plant Cell Environ. 23, 743–750 (2000).

    CAS  Google Scholar 

  5. Roumet, C. et al. Quantifying species composition in root mixtures using two methods: near-infrared reflectance spectroscopy and plant wax markers. New Phytol. 170, 631–638 (2006).

    CAS  Google Scholar 

  6. Tulloch,, A.P. Chemistry of waxes of higher plants. in Chemistry and Biochemistry of Natural Waxes (ed. Kolattukudy, P.E.) 235–287 (Elsevier, Amsterdam, Holland, 1976).

    Google Scholar 

  7. Dove, H. & Mayes, R.W. The use of plant wax alkanes as marker substances in studies of the nutrition of herbivores: a review. Aust. J. Agric. Res. 42, 913–952 (1991).

    CAS  Google Scholar 

  8. Dove, H. & Mayes, R.W. Plant wax components: a new approach to estimating intake and diet composition in herbivores. J. Nutr. 126, 13–26 (1996).

    CAS  Google Scholar 

  9. Dove, H. & Mayes, R.W. Using n-alkanes and other plant wax components to estimate intake, digestibility and diet composition of grazing/browsing sheep and goats. Small Ruminant Res. 59, 123–139 (2005).

    Google Scholar 

  10. Mayes, R.W., Dove, H., Chen, X.B. & Guada, J.A. Advances in the use of faecal and urinary markers for measuring diet composition, herbage intake and nutrient utilisation in herbivores. in Recent Developments in the Nutrition of Herbivores (eds. Journet, M., Farce, M.-H. & Demarqully, C.) 381–406 (INRA Editions, Paris, 1995).

    Google Scholar 

  11. Mayes,, R.W. & Dove,, H. The use of n-alkanes and other plant-wax compounds as markers for studying the feeding and nutrition of large mammalian herbivores. in Herbivores: The Assessment of Intake, Digestibility and the Roles of Secondary Compounds (eds. Sandoval-Castro, C.A., DeB Hovell, D.B., Torres-Acosta, J.F.J. & Ayala-Burgos, A.) 153–182 (BSAS Publication 34 (Nottingham, U.K.) 2006).

    Google Scholar 

  12. Oliván, M., Dove, H., Mayes, R.W. & Hoebee, S.E. Recent developments in the use of alkanes and other plant wax components to estimate herbage intake and diet composition in herbivores. Rev. Portuguesa Zootec. 6, 1–26 (1999).

    Google Scholar 

  13. Mayes, R.W. New potential markers for determining diet composition. in Proc. 9th Eur. Intake Workshop, 63–66 (IGER, North Wyke, Devon, UK, 1998).

    Google Scholar 

  14. Mayes, R.W., Lamb, C.S. & Colgrove, P.M. The use of dosed and herbage n-alkanes as markers for the determination of herbage intake. J. Agric. Sci. Camb. 107, 161–70 (1986).

    CAS  Google Scholar 

  15. Dove, H. Using the normal-alkanes of plant cuticular wax to estimate the species composition of herbage mixtures. Aust. J. Agric. Res. 43, 1711–1724 (1992).

    CAS  Google Scholar 

  16. Mayes, R.W. & Lamb, C.S. The possible use of n-alkanes in herbage as indigestible faecal markers. Proc. Nutr. Soc. 43, 39A (1984).

    Google Scholar 

  17. Malossini, F., Bovolenta, S., Piasentier, E., Piras, C. & Martillotti, F. Comparison of n-alkanes and chromium oxide methods for estimating herbage intake by grazing dairy cows. Anim. Feed Sci. Tech. 61, 155–165 (1996).

    CAS  Google Scholar 

  18. Dove, H., Freer, M. & Foot, J.Z. The nutrition of grazing ewes during pregnancy and lactation: a comparison of alkane-based and chromium/in vitro-based estimates of herbage intake. Aust. J. Agric. Res. 51, 765–777 (2000).

    CAS  Google Scholar 

  19. Mayes, R.W. & Duncan, A.J. New developments in the use of plant-wax markers to determine intake. in Emerging Techniques for Studying the Nutrition of Free Ranging Herbivores (eds. Dove, H. & Coleman, S.W.) CD-ROM (Vth International Symposium on the Nutrition of Herbivores, San Antonio, TX, April 1999).

  20. Dove, H., Freer, M. & Foot, J.Z. Alkane capsules for measuring pasture intake. Proc. Nutr. Soc. Aust. 13, 131 (1988).

    Google Scholar 

  21. Vulich, S.A., O'Riordan, E.G. & Hanrahan, J.P. Use of n-alkanes for the estimation of herbage intake in sheep: accuracy and precision of the estimates. J. Agric. Sci. Camb. 116, 319–323 (1991).

    CAS  Google Scholar 

  22. Giráldez, F.J., Lamb, C.S., López, S. & Mayes, R.W. Effects of carrier matrix and dosing frequency on digestive kinetics of even-chain alkanes and implications on herbage intake and rate of passage studies. J. Sci. Food Agric. 84, 1562–1570 (2004).

    Google Scholar 

  23. Letso, M. A Study of the Use of n-Alkanes to Determine Dietary Intake and Digestibility in Grazing Rabbits. MSc Thesis, University of Aberdeen, (1995).

  24. Dove, H. & Oliván, M. Using synthetic or beeswax alkanes for estimating supplement intake in sheep. Anim. Prod. Aust. 22, 189–192 (1998).

    Google Scholar 

  25. Sehested, J. et al. Use of n-alkanes to estimate grass intake and digestibility in sows. in Emerging Techniques for Studying the Nutrition of Free Ranging Herbivores (eds. Dove, H. & Coleman, S.W.) CD-ROM (Vth International Symposium on the Nutrition of Herbivores, San Antonio, TX, April 1999)..

  26. Elwert, C. & Dove, H. Estimation of roughage intake in sheep using a known daily intake of a labelled supplement. Anim. Sci. 81, 47–56 (2005).

    CAS  Google Scholar 

  27. Marais, J.P., Figenschou, D.L., Escott Watson, P.L. & Webber, L.N. Administration in suspension form of n-alkane external markers for dry matter intake and diet selection studies. J. Agric. Sci. Camb. 126, 207–210 (1996).

    CAS  Google Scholar 

  28. Hendricksen, R.E. et al. Using molasses as an alternative to controlled release devices for administering n-alkane markers to cattle. Anim. Sci. 76, 471–480 (2003).

    Google Scholar 

  29. Dove, H., Mayes, R.W., Lamb, C.S. & Ellis, K.J. Factors influencing the release rate of alkanes from an intraruminal controlled release device, and the resultant accuracy of intake estimation in sheep. Aust. J. Agric. Res. 53, 681–696 (2002).

    CAS  Google Scholar 

  30. Molle, G., Decandia, M. & Dove, H. A comparison between different procedures for dosing n-alkanes to sheep. in Proc. 9th Eur. Intake Workshop, 53–57 (IGER, North Wyke, Devon, UK, 1998).

    Google Scholar 

  31. France, J., Dhanoa, M.S., Siddons, R.C., Thornley, J.H.M. & Poppi, D.P. Estimating the production of faeces by ruminants from faecal marker concentrations. J. Theor. Biol. 135, 383–391 (1988).

    CAS  Google Scholar 

  32. Galyean, M.L. Technical note—an algebraic method for calculating fecal output from a pulse dose of an external marker. J. Anim. Sci. 71, 3466–3469 (1993).

    CAS  Google Scholar 

  33. Duncan, A.J., Mayes, R.W., Lamb, C.S., Young, S.A. & Castillo, I. The use of naturally occurring and artificially applied n-alkanes as markers for estimation of short-term diet composition and intake in sheep. J. Agric. Sci. Camb. 132, 233–246 (1999).

    CAS  Google Scholar 

  34. Giráldez, F.J., López, S., Lamb, C.S. & Mayes, R.W. The use of even-chain alkanes sprayed onto herbage as rate of passage markers in goats. Livestock Sci. 100, 195–202 (2006).

    Google Scholar 

  35. Mayes, R.W., Giráldez, F.J. & Lamb, C.S. Estimation of gastrointestinal passage rates of different plant components in ruminants using isotopically-labelled plant wax hydrocarbons or sprayed even-chain alkanes. Proc. Nutr. Soc. 56, 187A (1997).

    Google Scholar 

  36. Mayes, R.W., Lamb, C.S. & Colgrove, P.M. Digestion and metabolism of dosed even-chain and herbage odd-chain n-alkanes in sheep. in Proc. 12th Gen. Meeting Eur. Grasslands Fed., 159–163 (1988).

    Google Scholar 

  37. Hameleers, A. & Mayes, R.W. The use of n-alkanes to estimate herbage intake and diet composition by dairy cows offered a perennial ryegrass/white clover mixture. Grass Forage Sci. 53, 164–169 (1998).

    CAS  Google Scholar 

  38. Dove, H. & Moore, A.D. Using a least-squares optimisation procedure to estimate botanical composition based on the alkanes of plant cuticular wax. Aust. J. Agric. Res. 46, 1535–1544 (1995).

    CAS  Google Scholar 

  39. Newman, J.A., Thompson, W.A., Penning, P.D. & Mayes, R.W. Least-squares estimation of diet composition from n-alkanes in herbage and faeces using matrix mathematics. Aust. J. Agric. Res. 46, 793–805 (1995).

    CAS  Google Scholar 

  40. Mayes, R.W. et al. Novel approaches to the estimation of intake and bioavailability of radiocaesium in ruminants grazing forested areas. Sci. Total Environ. 157, 289–300 (1994).

    CAS  Google Scholar 

  41. Salt, C.A., Mayes, R.W., Colgrove, P.M. & Lamb, C.S. The effects of season and diet composition on the radiocaesium intake by sheep grazing a heather moorland. J. Appl. Ecol. 31, 125–136 (1994).

    CAS  Google Scholar 

  42. Hameleers, A. & Mayes, R.W. The use of n-alkanes to estimate supplementary grass silage intake in grazing dairy cows. J. Agric. Sci. Camb. 131, 205–209 (1998).

    CAS  Google Scholar 

  43. Dillon, P., Crosse, S., O'Brien, B. & Mayes, R.W. The effect of forage type and level of concentrate supplementation on the performance of spring-calving dairy cows in early lactation. Grass Forage Sci. 57, 212–223 (2002).

    CAS  Google Scholar 

  44. Bugalho, M.N., Mayes, R.W. & Milne, J.A. The effects of feeding selectivity on the estimation of diet composition using the n-alkane technique. Grass Forage Sci. 57, 224–231 (2002).

    CAS  Google Scholar 

  45. Coleman, S.W., Christiansen, S. & Shenk, J. Prediction of botanical composition using NIRS calibrations developed from botanically pure samples. Crop Sci. 30, 202–207 (1990).

    Google Scholar 

  46. Walker, J.W., McCoy, S.D., Launchbaugh, K.L., Fraker, M.J. & Powell, J. Calibrating fecal NIRS equations for predicting botanical composition of diets. J. Range Manage. 55, 374–382 (2002).

    Google Scholar 

  47. André, J. & Lawler, I.R. Near infrared spectroscopy as a rapid and inexpensive means of dietary analysis for a marine herbivore, dugong Dugong dugon. Mar. Ecol. Prog. Ser. 257, 259–266 (2003).

    Google Scholar 

  48. Fulford,, G. The Potential Use of Plant Hydrocarbons Additional to n-Alkanes as Markers for Estimating Diet Composition in Large Herbivores. MSc Thesis, University of Aberdeen, (1994).

  49. Dove, H. & Oliván, M. The possible use of the alkenes (unsaturated hydrocarbons) of plant cuticular wax as diet composition marker in sheep. in Herbivores: The Assessment of Intake, Digestibility and the Roles of Secondary Compounds (eds. Sandoval-Castro, C.A., DeB Hovell, D.B., Torres-Acosta, J.F.J. & Ayala-Burgos, A.) 1–7 (BSAS Publication 34 (Nottingham, U.K.) 2006).

    Google Scholar 

  50. Ali, H.A.M. The Potential Use of Some Plant Wax Compounds as Faecal Markers to Measure the Botanical Composition of Herbivore Diets. PhD Thesis, University of Aberdeen, (2003).

  51. Ali, H.A.M. et al. 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. J. Agric. Sci. Camb. 142, 71–78 (2004).

    CAS  Google Scholar 

  52. Bugalho, M.N., Dove, H., Kelman, W., Wood, J.T. & Mayes, R.W. Plant wax alkanes and alcohols as herbivore diet composition markers. J. Range Manage. 57, 259–268 (2004).

    Google Scholar 

  53. Ali, H.A.M. et al. The potential of long-chain fatty alcohols and long-chain fatty acids as diet composition markers. J. Agric. Sci. Camb. 143, 85–95 (2005).

    CAS  Google Scholar 

  54. Grace, N.D. & Body, D.R. The possible use of long-chain (C19–C32) fatty acids in herbage as an indigestible faecal marker. J. Agric. Sci. Camb. 97, 743–745 (1981).

    CAS  Google Scholar 

  55. Ali, H.A.M., Mayes, R.W., Hector, B.L. & Ørskov, E.R Assessment of n-alkanes, long-chain fatty alcohols and long-chain fatty acids as diet composition markers: the concentrations of these compounds in rangeland species from Sudan. Anim. Feed Sci. Technol. 121, 257–271 (2005).

    CAS  Google Scholar 

  56. Jayakody, J.A.D.S.S. A Study of the Effects of Human Disturbance on Habitat Use, Behaviour and Diet Composition in Red Deer (Cervus elaphus L.). PhD Thesis, University of Aberdeen, (2005).

  57. Brewer, M.J. et al. A hierarchical model for compositional data analysis. J. Agric. Biol. Environ. Stat. 10, 19–34 (2005).

    Google Scholar 

  58. Dove, H., Scharch, C.P., Oliván, M. & Mayes, R.W. Using n-alkanes and known supplement intake to estimate roughage intake in sheep. Anim. Prod. Aust. 24, 57–60 (2002).

    Google Scholar 

  59. Dove, H., Charmley, E. & Kleven, K. Using n-alkanes to estimate intakes of mixed forages by feeding a known amount of an alkane-labelled supplement. Can. J. Anim. Sci. 83, 641–642 (2003).

    Google Scholar 

  60. Elwert, C., Dove, H. & Rodehutscord, M. Effect of roughage species on faecal alkane recovery in sheep, and the effect of drying treatment on alkane concentration. Aust. J. Exp. Agric. 46, 771–776 (2006).

    CAS  Google Scholar 

  61. Christie, W.W. Lipid Analysis 3rd edn. (Oily press, Bridgwater, UK, 2002).

  62. Burdge, G.C., Wright, P., Jones, A.E. & Wotton, S.A. A method for separation of phosphatidylcholine, triacylglycerol, non-esterified fatty acids and cholesterol esters from plasma by solid-phase extraction. Br. J. Nutr. 84, 781–787 (2000).

    CAS  Google Scholar 

  63. Oliván, M. & Osoro, K. Effect of temperature on alkane extraction from faeces and herbage. J. Agric. Sci. Camb. 132, 305–312 (1999).

    Google Scholar 

  64. Christie, W.W. Silver ion chromatography using solid-phase extraction columns packed with a bonded-sulfonic acid phase. J. Lipid Res. 30, 1471–1473 (1989).

    CAS  Google Scholar 

  65. Carlson, D.A., Roan, C.-S., Yost, R.A. & Hector, J. Dimethyl disulfide derivatives of long chain alkenes, alkadienes and alkatrienes for gas chromatography/mass spectrometry. Anal. Chem. 61, 1564–1571 (1989).

    CAS  Google Scholar 

  66. Hulbert, I.A.R., Iason, G.R. & Mayes, R.W. The flexibility of an intermediate feeder: dietary selection by mountain hares measured using faecal n-alkanes. Oecologia 129, 197–205 (2001).

    Google Scholar 

  67. Rao, S.J., Iason, G.R., Hulbert, I.A.R., Mayes, R.W. & Racey, P.A. Estimating diet composition for mountain hares in newly established native woodland: development and application of plant-wax faecal markers. Can. J. Zool. 81, 107–1056 (2003).

    Google Scholar 

  68. Mayes, R.W., Iason, G.R., White, N. & Palo, T. Measuring diet composition and food intake by moose in the Swedish boreal forest: integrating GPS and faecal marker technologies. in Tracking Animals with GPS (eds Sibbald, A.M. & Gordon, I.J.) 77–80 (Macaulay Institute, Aberdeen, Scotland, 2001).

    Google Scholar 

  69. Hatt, J.-M., Lechner-Doll, M. & Mayes, B. The use of dosed and herbage n-alkanes as markers for the determination of digestive strategies of captive giraffes (Giraffa camelopardalis). Zool. Biol. 17, 295–309 (1998).

    Google Scholar 

  70. Wilson, H., Sinclair, A.G., Hovell, F.DeB., Mayes, R.W. & Edwards, S.A. Validation of the n-alkane technique for measuring herbage intake in sows. Proc. Braz. Soc. Anim. Sci., 171 (1999).

  71. O'Keefe, N.M. & McMeniman, N.P. The recovery of natural and dosed n-alkanes from the horse. Anim. Prod. Aust. 22, 37 (1998).

    Google Scholar 

  72. Ordakowski, A.L. et al. Alkanes as internal markers to estimate digestibility of hay or hay plus concentrate diets in horses. J. Anim. Sci. 79, 1516–1522 (2001).

    CAS  Google Scholar 

  73. Martins, H., Elston, D.A., Mayes, R.W. & Milne, J.A. Assessment of the use of n-alkanes as markers to describe the complex diets of herbivores. J. Agric. Sci. Camb. 138, 425–434 (2002).

    CAS  Google Scholar 

  74. Hatt, J.-M., Mayes, R.W., Clauss, M. & Lechner-Doll, M. Use of artificially applied n-alkanes as markers for the estimation of digestibility, food selection and intake in pigeons (Columba livia). Anim. Feed Sci. Technol. 94, 65–76 (2001).

    CAS  Google Scholar 

  75. Hatt, J.-M. et al. The use of dosed and herbage n-alkanes as markers for the determination of intake, digestibility, mean retention time and diet selection in Galapagos tortoises (Geochelone nigra). Herpetol. J. 12, 45–54 (2002).

    Google Scholar 

  76. Gudmundsson, O. & Halldorsdottír, K. The use of n-alkanes as markers for determination of intake and digestibility of fish feed. J. Appl. Ichthyol. Z. Angew. Ichthyol. 11, 354–358 (1995).

    CAS  Google Scholar 

  77. Stephen, L., Benton, T.G., Bryant, D.M. & Mayes, R.W. Remote assessment of wild bird diet: an investigation into current and novel tools for dietary estimation. in 90th Annual Meeting of the Ecological Society of America (ESA) and IX Montreal INTECOL congress 7–12th August [abstracts CD-ROM] (2005).

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Supplementary protocol for the formation of trimethylsilyl (TMS) derivatives of LCOH (PDF 91 kb)

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Dove, H., Mayes, R. 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. Nat Protoc 1, 1680–1697 (2006). https://doi.org/10.1038/nprot.2006.225

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