polymerized phenolic compounds, lignin
should theoretically be indigestible and
suitable as an internal marker. However,
lignin has a complex structure, the
chemical properties of which may vary
from plant to plant and even from one
plant part to another. A shortcoming of
lignin as a marker is its low and inconsis-
tent recovery in the faeces. Furthermore,
lignin can give positive faecal recoveries
by binding to components of indeter-
minate nature (Thewis et al., 1989). Acid
detergent lignin recoveries of between 0.52
and 1.16 were found by Cochran et al.
(1986).
Acid detergent lignin is determined
gravimetrically after cellulose has been
removed from ADF by treatment with 26M
sulphuric acid and the ash content has
been deducted (Van Soest, 1963). Alkaline
peroxide lignin, a less digestible lignin
fraction, prepared by treating the sample
with alkaline hydrogen peroxide before the
acid detergent step in the acid detergent
lignin procedure, was proposed as inter-
nal marker for digestibility predictions.
However, Momont et al. (1994) showed
that although alkaline peroxide lignin was
nearly totally recoverable from the faeces,
the results on mature prairie grass hay
were variable and adversely affected dry
matter intake predictions. It was concluded
that alkaline peroxide lignin does not
appear to improve the accuracy of predict-
ing the dry matter digestibility of grasses
over acid detergent lignin (Sunvold and
Cochran, 1991).
Insoluble ash
Acid-insoluble ash is often used as an
indigestible marker for estimating apparent
metabolizable energy in chickens and
digestibility in ruminants. It is prepared by
drying and ashing the sample, and boiling
the ashed sample in 2Mhydrochloric acid
for 5 min. The ash content is determined
gravimetrically after the hot hydrolysate
has been filtered, washed free of acid and
re-ashed (Van Keulen and Young, 1977).
Contamination of feed and faeces samples
with dust or soil could lead to erroneous
results.
Odd-chain n-alkanes
Plant n-alkanes are simple straight-chain
hydrocarbons with carbon chain lengths
ranging from pentacosane (C 25 ) to penta-
triacontane (C 35 ). They are components of
the cuticular wax layer covering all higher
plants and occur in plant material mainly
as odd-chain molecules. Individual alkanes
in plants differ in concentration, resulting
in each plant having a unique alkane
profile, with hentriacontane (C 31 ) and
tritriacontane (C 33 ) usually as the major
components (Table 12.1). Alkanes are
associated predominantly with the
particulate matter in digesta, and some are
virtually quantitatively excreted in the
faeces. Their stability in the digestive tract
of the ruminant appears to increase with
the size of the molecule. The recovery in
the faeces of C 33 is about 0.87, while that of
C 35 has been estimated at 0.95. Alkanes
appear to be completely stable in the
digestive tract of mountain hares. Natural
alkanes are used to estimate digestibility
and species selection by the grazing
animal.
Separation of alkanes from plant or
faeces samples involves saponification in
alcoholic potassium hydroxide and extrac-
tion with non-polar solvents such as
heptane (Dillon and Stakelum, 1990). The
extracted alkanes are purified by means of
a silica gel column, and the alkanes are
separated and quantified by means of
capillary gas chromatography (Dove and
Mayes, 1991).2,6-Diaminopimelic acid (DAPA)
DAPA is an amino acid occurring in varying
concentrations in bacterial cells. Since the
DAPA to protein ratios of mixed ruminal
bacteria are relatively constant, it has
become the most commonly used internal
marker for estimating microbial protein syn-
thesis (Czerkawski, 1974). Broderick and
Merchen (1992) pointed out that this marker
has many serious shortcomings. Because
DAPA only occurs in microbial cell walls,
the DAPA to protein ratio could change
depending on microbial cell size and shape.
Of greater importance is the occurrence of
substantial amounts of DAPA in certain feedUse of Markers 257