strained through cheesecloth into a prewarmed 
insulated thermos (1-2 gallons depending upon 
need). The thermos is completely filled to elim- 
inate air. Several subsamples are taken from 
different parts of the rumen. When available, 
several animals are sampled and the rumen liq- 
uor mixed. The inoculum-source cattle are 
trapped at dusk, held off feed overnight, and 
sampled the following morning. Alexander and 
McGowan (1966) illustrated the small varia- 
tion in activity of rumen liquor due to the ef- 
fect of inoculum extraction related to feeding. 
They also demonstrated no real differences in 
rumen inoculum activity related to animal var- 
iances. Therefore, inoculum could be used from 
a single animal or as a mixture from several 
animals. 
The in vitro digestion laboratory procedure 
is as follows: 
Step I: 
1. Grind dried forage samples to pass a 0.5 
mm. screen. Redry ground samples for 4 hours 
at 100° C. 
2. Place triplicate 0.5-gram (+0.1 mg.) sam- 
ples into 100-ml. digestion tubes. Include 
empty reagent check tubes in each digestion 
trial. 
3. Saturate buffer solution with carbon diox- 
ide to pH 6.8 by bubbling, and warm to 38.5° 
C 
4, Add whole rumen fluid strained through 
cheesecloth to buffer solution (one part rumen 
fluid to four parts buffer solution). 
5. Add rumen fluid-buffer solution mixture 
to digestion tubes containing forage samples 
(50 ml./tube). 
6. Immediately exhaust air in remaining 
space above the liquid in the tube with carbon 
dioxide, and insert closure. The closures are 
stoppers fitted with an exhaust valve to permit 
FIGURE 3.—Water bath used for incubation during an 
in vitro fermentation trial. 
escape of gases within the tube during fermen- 
tation, and yet prevent reentry of air into the 
tube (Tilley and Terry 1963). 
7. Incubate 48 hours at 38.5° C. (fig. 3). 
8. Shake tubes individually to mix forage 
initially and 2, 6, and 24 hours after start of 
incubation. 
Step IT: 
1. Add 4 ml. of HCl solution to reduce to 
pH 1-2 (acid added in four 14-ml. doses and 
two 1-ml. doses to prevent excess foaming). 
2. Add 5 ml. of pepsin solution and close 
tubes. 
3. Incubate 48 hours at 38.5° C. 
4, Shake tubes individually to mix forage 
initially and 2, 6, and 24 hours after the start 
of incubation. 
5. Add 12 ml. of sodium carbonate solution 
to stop enzymatic action.’ 
6. Vacuum filter,* dry, and weigh. 
Since many digestion tubes may be used 
simultaneously and since filtration may extend 
over a long period step 5 has been added to 
stop enzymatic action in all tubes at the same 
time. Pepsin becomes inactive at about pH 4.6, 
and is readily denatured at pH value higher 
than 6; optimum activity occurs at approxi- 
mately pH 2 (Fruton and Simmonds 1958). Di- 
gestibility is similar whether the substrate is 
filtered immediately following digestion, or 
whether it is delayed after the addition of so- 
‘This step is optional. Whether it is taken depends 
on the number of tubes to be filtered 
*Grade GFA Whatman glass fiber papers (fiber di- 
ameter < 1 micron) are used. 
DIGESTIBILITY (percent) 
SODIUM CARBONATE AND FILTER 
20 30 40 50 60 
DIGESTIBILITY (percent) 
IMMEDIATE FILTER 
FIGURE 4.—Comparison of immediate filter after in 
vitro digestion and delayed filter after adding sodium 
carbonate to stop enzymatic action. 
89 
