Farm Animal Metabolism and Nutrition

to measuring in vitrogas production for
feedstuff evaluation. The early Menke
syringe method is still used in many parts
of the world, most notably at Hohenheim,
Germany, its birthplace (Blümmel et al.,
1997a). Computer-linked electronic pressure
sensors have been applied in two different
ways. In the first of these, gas is allowed to
accumulate in the digestion vessel and
changes in gas pressure are recorded (Pell
and Schofield, 1993). In the second, gas is
vented at intervals determined by the pres-
sure sensor and recorded by the computer.
The volume/time profile is then calculated
from the venting data.

Factors Affecting Gas Measurements

Before attempting to evaluate the respec-
tive merits and demerits of these different
approaches, let us step back for a moment
and consider some of the factors that must
be taken into account in any protocol for
gas measurement.

Sample size, physical state

As we have seen, the gas volume
produced by complete digestion of 1 g of
fibre is about 350 ml. Digestibility (rather
than digestion rate) measurements will
therefore require the use of either small
samples or equipment able to handle
large gas volumes. Many early studies
using the syringe method were concerned
with comparing ratesof gas production
from different substrates and, therefore,
did not have to deal with large gas
volumes. Gas volumes are proportional to
sample size.
Because many animal feedstuffs are
not homogeneous, they present sampling
problems. These problems can be reduced
by grinding the feedstuff. However, we
must also remember that the digestion
rates we measure in vitro may depend
upon particle size. This dilemma is not
peculiar to the gas technique but underlies
any in vitromethod involving an insoluble
substrate. In practice, the usual approach

is to grind the feedstuff to pass a 1 mm

sieve, to ferment multiple samples and to

discount the particle size effect on the

grounds that chewing and rumination in

the animal will produce a result similar to

grinding.

Ruminal fluid (RF)

There are two questions to be answered

here, namely pre-treatment and amount.

RF contains both the microorganisms

(bacteria, fungi, protozoa) and soluble

factors (VFAs, vitamins) needed for fibre

digestion. The composition of this fluid

will vary from day to day and from animal

to animal, and these variations may affect

in vitro digestion profiles. One way to

reduce this variation is to filter the fluid

through glass wool, pellet the micro-

organisms by centrifugation and resuspend

them in a defined medium (Doane et al.,

1997b).

Ruminal fluid is usually diluted with a

buffered salt solution before use. Previous

in vitrostudies (Hungate et al., 1955; Tilley

and Terry, 1963) showed that a buffered

medium containing 20–25% RF gave the

best results. A higher content of RF will

produce higher blanks (samples incubated

with RF but without substrate).

pH control

The standard buffer has been based on

McDougal’s analysis of sheep saliva

(McDougal, 1949) and contains both

bicarbonate and phosphate. Some varia-

tions on the exact buffer composition are

seen among different laboratories. The

primary buffering agent, bicarbonate, is

consumed as VFAs are released during

digestion and thus the pH declines. For

quantitative gas measurements using this

buffer, it is important that the pH be held

within the range 6.8–6.2. At a lower pH,

the cellulolytic bacteria become less active

(Russell and Dombrowski, 1980) and the

yield of CO 2 per mole of VFAs declines

(Beuvink and Spoelstra, 1992).

Gas Production Methods 213