Inorganic^15 nitrogenInorganic^15 N is often preferred to other
isotopic markers for estimating microbial
nitrogen synthesis since it is a stable
isotope and does not present an environ-
mental hazard. In order to obtain a uniform
distribution of^15 N marker throughout the
microbial nitrogen pools, 10–85%^15 N-
enriched ammonium sulphate is infused
continuously through the rumen fistula for
a period of 48 h. After this period, there is
little difference between the^15 N enrich-
ment in ruminal and post-ruminal
microbial pools sampled simultaneously,
although it takes 7–8 days to obtain a
plateau and constant specific enrichment
of ruminal or post-ruminal nitrogen pools.
If the^15 N is administered by including it in
the feed, a feeding period of longer than
48 h is required (Broderick and Merchen,
1992).
Procedures for quantifying^15 N have
been reviewed (International Atomic Energy
Agency, 1985). Although the accuracy and
precision of analysis by isotope ratio mass
spectrometry is very good (enrichment of
0.01–0.05% is adequate for most studies),
the procedure is tedious, as the nitrogen
fraction under study must be isolated prior
to analysis.
L-[4,5-^3 H]LeucineL-[4,5-^3 H]Leucine, infused into the rumen
for 36 h, is incorporated into the protein
fraction of all microbial species, and
presents an accurate means of estimating
microbial nitrogen flow (Sinclair et al.,
1993). [^3 H]Leucine is measured by liquid
scintillation counting.
Inorganic^35 sulphurLabelled sulphur can be infused con-
tinuously (14 days) into the rumen in the
form of^35 S-labelled sodium sulphate
(Kang-Meznarich and Broderick, 1981).
After reduction to sulphide, the label is
incorporated into bacterial protein by de
novosynthesis of the sulphur-containing
amino acids, cysteine and methionine, or
by incorporation into substances such as
coenzyme A. Protozoa eventually become
labelled by indirect means. Collected
digesta samples are treated and analysed
according to Elliott and Armstrong (1982).
Although of relatively short half-life (87
days),^35 S is a potential health hazard and
may accumulate in meat and milk, render-
ing it unsuitable for consumption.Inorganic^32 phosphorusInorganic^32 P has also been used as a
microbial marker by incorporating it into
microbial phospholipids (Bucholtz and
Bergen, 1973), but substantial precautions
need to be taken for its use as a radioactive
tracer.Radionuclide-labelled microspheresThe microspheres are commercially avail-
able polymeric resin-covered, non-bio-
degradable, ion exchange beads labelled
with^141 Ce,^57 Co,^95 Nb or^103 Ru (Young et
al., 1991). When administered directly into
the rumen of sheep, the microspheres are
injected as a dextran suspension. However,
when fed to sheep, they are blended with
finely ground fish meal and pelleted. The
radionuclide content is determined in
oven-dried samples of faeces compressed
into cylinders and read directly in a
neckless counting vial in a gamma counter.
Microspheres were shown not to pass
through the wall of the gastrointestinal
tract and gave a satisfactory recovery of
98.6% in the faeces (Young et al., 1991).
Microspheres do not represent the flow of
any particular food particle in the digestive
tract, but appear to be suitable for compara-
tive studies. They have the advantages of
uniformity of particle size and density
which do not change during passage
through the gut. They are available with
different radionuclide labelling and are
measured easily in faeces.Use of Markers 263