The inoculum should be kept in anaerobic 
conditions throughout the digestion process. 
This is generally accomplished by one of two 
techniques: (1) continuous flushing with car- 
bon dioxide or some other suitable gas during 
the entire digestion period (Van Dyne 1962) or 
(2) initial flushing with gas and closure of the 
digestion tubes with closures that provide for 
gas release (Tilley and Terry 1963). In the lat- 
ter method, the initial flushing to displace the 
oxygen prior to closure is essential for maxi- 
mum fermentation (table 2). 
Length of Fermentation 
Fermentation time varies among individual 
laboratories; most universal time lengths are 
12, 24, and 48 hours fermentation by the mi- 
cro-organisms; however, many other fermenta- 
tion times have been explored. Differences in 
digestion have been significant, especially in 
the shorter digestion times. Dry-matter diges- 
tion increased with increasing length of fer- 
mentation up to the 48 hours tested in our lab- 
oratory (fig. 1). Similar results have been re- 
ported in other laboratories. Addition of acid- 
pepsin solution will increase digestion for vari- 
ous lengths of time. Tilley et al. (1960) used 48 
hours of acid-pepsin digestion, while Barnes 
(1966) used 24 hours. Differences in digestibil- 
ity between these two time lengths are small 
(table 3), therefore either time length can be 
used effectively. 
60 
50 
40 
30 
DIGESTIBILITY (percent) 
20 
Cercocarpus breviflorus 
eg Sitanion hystrix 
Quercus turbinella 
esate? ee So Eragrostis curvula 
Bouteloua gracilis 
10) 12 24 48 96 
—=— MICROORGANISM ———*—*—— ACID-PEPSIN ———> 
DIGESTION TIME (hours) 
FIGURE 1.—Rates of in vitro dry matter digestion with 
rumen inoculum, plus additional acid-pepsin digestion. 
TABLE 38.—Percent forage digestibility when 
using 24 and 48 hours acid-pepsin digestion in 
the two-stage technique (Tilley and Terry 
1963). Forages were collected from the chap- 
arral range during November 1966 
Hours 
Forage 48 24 
Quercus turbinella (stems) ______ 24.0 23.4 
Ceanothus greggii (stems) ______ 24.3 25.8 
Cercocarpus breviflorus (stems) _ 32.5 82.3 
Rhus trilobata (stems) _______- 41.5 37.7 
Q. turbinella (leaves) __________ 47.7 44,5 
C. greggti (leaves) ____________- 44.5 45.0 
C. breviflorus (leaves) __________ 54.0 51.5 
Diet mixture (steer 6)? 37.6 36.0 
Diet mixture (steer 4) __________ 51.0 48.4 
Sitanion hystrix ______________ S12 56.9 
Bouteloua curtipendula _________- 41.7 40.4 
Eragrostis lehmanniana _______- 5 Be 48.3 
PU SCUT UU te: Bees i. Met med T Digas-| 43.0 41.0 
Bie TAGSUED Jeers Uae in ee ene 46.7 44.9 
Be OVUCWis. = yo Bees. Joes: 44.5 42.2 
141 0 aa eee cl 42.8 41.2 
*Diet mixture refers to forages removed from the 
rumen of cattle in rumen evacuation trials. 
Forage digestibilities obtained with various 
lengths of micro-organism fermentation have 
been attributed to different aspects of nutri- 
tion. For instance, digestibilities with short pe- 
riods of fermentation appear related to the 
rate of voluntary intake by animals, while long 
periods describe forage digestibility.t The lev- 
eling off of rumen micro-organism activity 
with long periods of fermentation, which de- 
scribes forage digestibility, has been attributed 
to the effect of lignin. Apparently lignin in for- 
ages is a physical barrier to the rumen micro- 
organisms.°® 
Digestion Components 
Various components in forage are known to 
be (1) water soluble, (2) digested by micro-or- 
ganisms, or (8) digested by acid-pepsin. For- 
age samples are processed by three methods to 
ascertain the amounts of each of these three 
components. The water-soluble portion is dis- 
solved in buffer solution for 48 hours; diges- 
tion for 48 hours in buffer solution plus rumen 
inoculum dissolves those portions of the forage 
that are water soluble or digestible by micro- 
organisms; and further digestion in acid-pep- 
sin for 48 hours dissolves that portion digested 
by acid-pepsin. Average in vitro dry matter di- 
gestibilities for chaparral forages collected in 
May were: Water soluble, 20.9 percent; mi- 
cro-organism digested, 21.9 percent; and acid- 
pepsin digested, 7.0 percent. Thus, total digest- 
*Barnes 1966; Crampton 1957; Crampton et al. 
1960; Donefer et al. 1960. 
*Dehority and Johnson 1961; Dehority et al. 1962; 
Kamstra et al. 1958; Sullivan 1959. 
87 
