ibility was 49.8 percent (table 4). Although 
variations due to season, location, and species 
would be expected, similar evaluations and re- 
sults were reported in Indiana by Barnes 
(1966). 
Digestibilities determined with micro-organ- 
isms and micro-organisms plus acid-pepsin 
were highly correlated (r=0.971, p<.01), 
which indicates the interchangeable predicting 
value of either method. Digestibilities deter- 
mined only with buffer were not significantly 
correlated with digestibilities from either of 
the other methods; consequently, the water- 
soluble component in forage apparently has no 
value in predicting forage digestibility. 
TABLE 4.—Comparison of percent in vitro dry 
matter digestibility methods, determined on 
chaparral forage collected during May 1966 
Methods 
ut 
Forage - #82 
Buffer Buffer+ , ,.29o 
micro- 29 § i 
: ae} 
organisms 5.4 0-5 
MEos 
Grasses: 
Bouteloua curti- 
pendula ______-._- 15.2 35.4 41.8 
B. gracilis ________- 17.0 42.0 47.6 
B. hirsuta ________- 14.7 38.8 46.2 
Eragrostis curvula __ 14.2 40.7 49.2 
E. lehmanniana ____ 16.1 43.3 54.3 
Sitanion hystrix ____ 21.4 59.8 69.5 
Brush: 
Cercocarpus 
breviflorus ______- 24,9 41.0 46.0 
Ceanothus greggti __ 22.0 36.1 41.6 
Quercus turbinella __ 30.5 38.4 45.7 
Rhus trilobata _____- 33.9 43.5 49.9 
Diet mixtures 
Steer 4 ___________- 21.3 44.8 49.1 
Steer 6 ______._ __- 19.2 50.8 57.0 
Mean ese f eee os 20.9 42.8 49.8 
A Standard Method 
In vitro digestion is a relatively new tool, 
and several techniques are used at various lab- 
oratories. One standard method of relating 
data from different laboratories is urgently 
needed. At the Tenth International Grassland 
Congress in Finland, several papers were pre- 
sented concerning in vitro digestion.® All these 
papers indicated use of a two-stage technique 
described by Tilley and Terry (1963), either as 
standard procedure or for comparison. Barnes 
(1966) and Tilley et al. (1960) indicated that 
the two-stage technique is the most reliable 
method for estimating in vivo forage digest- 
ibility. Van Soest (1967) also considered this 
°Barnes 1966; Dent and Aldrich 1966; Noller et al. 
1966; Van Soest et al. 1966; Wedin et al. 1966. 
88 
technique superior to other in vitro techniques 
because it involves essentially an enzymatic 
preparation of undigested cell walls. In appli- 
cation, the two-stage technique has been used 
to relate digestible forage consumed to beef 
production (Pearson 1967b). Although many 
scientists apparently think this technique ap- 
proaches the answer as a standard method, it 
was not included in collaborative studies to 
investigate possibilities for a standard method 
(Barnes 1967). Perhaps these collaborative 
studies were initiated prior to knowledge of the 
value of the two-stage technique. 
In Vitro Techniques Used at Flagstaff, 
Ariz. 
Since the two-stage in vitro method is fre- 
quently employed, and is the method used in 
our laboratory, the procedure used, with vari- 
ous modifications, will be outlined here. 
The equipment and reagents used include: 
Equipment 
Forage grinder or mill 
Reagents 
Buffer solution: 
Drying oven Ingredient g/liter H:O 
Analytical balance NaHCO 9.80 
Na-HPO, 2 7H:0 7.00 
Thermos KCl 57 
. NaCl AT 
Digestion containers MgSO, - 7H.0 12 
(100-ml. centrifuge CaCl. .04 
tubes). 
Incubator (water bath) Carbon dioxide gas 
pH meter Hydrochloric acid solution 
(one part HCl) (11.6N); 
four parts H.0) 
Sodium carbonate (53 g/liter 
Vacuum filter apparatus 
Automatic pipetter 
20) 
Pepsin (1:10,000) 0.12 g/5 
Whole rumen liquor is removed from ru- 
men-fistulated cattle (fig. 2) grazing the range 
forages to be evaluated. The rumen ingesta is 
FIGURE 2.—Rumen fluid for in vitro digestion trials is 
obtained from fistulated cattle. 
