sheep (Mehrez and Ørskov, 1977), but the
DM disappearance after ruminal incuba-
tion varies according to the location of the
bags in the rumen (Stritzler et al., 1990).
Stritzler et al.(1990) reported an increase
of disappearance in response to increased
string length, in relation to increased
microbial activity measured by the ATP
concentration within the bags. These varia-
tions are related to the stratification of
digesta and the variations in the concentra-
tion of microorganisms with sampling site,
dorsal and ventral sac.
The bag incubation sequence can also
influence digestion rates. Nocek (1985)
compared two bag incubation sequences:
all bags were placed in the rumen at once
and removed at designated time intervals,
or bags were introduced in reverse
sequence and removed all at once. The
former procedure resulted in lower varia-
tion and slower digestion rates, perhaps
because the digestion process is inter-
rupted when bags are removed then
replaced in the rumen. However, if the bags
are not introduced at the same time in the
rumen, they will not be submitted to the
same degradation conditions. In most
studies, animals are fed in two meals per
day, and the rumen environment cannot be
considered as stable. After feeding, the
rapidly changing rumen conditions such as
osmotic pressure, entrance of oxygen or
dilution and passage rate involve diurnal
changes in the microbial ecosystem (see
review by Dehority and Orpin, 1988) and
in its enzyme activity (Martin et al., 1993;
Fig. 11.1B). To compare degradation rates,
it is probably better to introduce all the
bags at the same time in the rumen, but
Huntington and Givens (1997) did not
report an effect of incubation sequence on
DM degradability of feeds.
Microbial colonization of bag samples:
input of microorganisms
The pore size and open area of the cloth
used for in situbags needs to be such that
the cloth permits the influx of digesting
agents and buffers. From the synthesis of
Huntington and Givens (1995), researchers
have favoured the use of two distinct
aperture ranges, <15 and >35 μm, and this
suggests two approaches to the problem:
those authors who are looking for minimiza-
tion of loss of undegraded particles, and
those who favour equating the internal
microbial microenvironment of the bag
with that of the rumen.
A more diverse microbial colonization
of feed samples is indicated in 40 μm as
compared with 20 μm bags, and may relate
to increased numbers of protozoa (Lindberg
et al., 1984) entering the bag. Meyer and
Mackie (1986) showed that maximum
influx of protozoa occurred after 12 h (50%
after 4 h) of rumen incubation. The number
of protozoa increases as the pore size of the
bags increases (Huntington and Givens,
1995). Even when pore size is sufficient for
protozoal influx, the bacterial population
inside the bag differs from that of the
surrounding rumen digesta, and more
particularly the cellulolytic population
(Meyer and Mackie, 1986). Therefore,
the fibrolytic activity of solid-adherent
microorganisms is lower in bag residues
than in rumen digesta (Nozière and
Michalet-Doreau, 1996). These differences
can be explained by the shorter time spent
in the rumen by the bag particles, the lack
of mastication of incubated forages and the
confining conditions of feed inside the bag.
In bags, the possibilities of exchange
between the feed particles and the rumen
medium are limited by the surface area of
the pores in the bag material (Lindberg et
al., 1984), which accounts for the acidifica-
tion of the medium inside the bags. The pH
in the bags is lower than the pH of the
rumen contents, at least for the longest
incubation time (Nozière and Michalet-
Doreau, 1996), and this difference may be
due to an accumulation of volatile fatty
acids inside the bags (Marinucci et al.,
1992). The amylolytic enzyme activity of
solid-adherent microorganisms is higher in
bags containing barley than in bags con-
taining maize, at least for the first hours of
incubation (Nozière and Michalet-Doreau,
1997). The amylase activity of micro-
organisms colonizing cereals in the bags
would depend on the nature of the sub-
strate in the bag, and more specifically on238 P. Nozière and B. Michalet-Doreau