Digestibility Trials: In Vitro Techniques 
HENRY A. PEARSON?! 
Many tons of vegetation are produced and 
converted to beef, mutton, wool, or venison 
each year on rangelands throughout the United 
States. To properly manage these ranges, we 
measure production, utilization, nutritive 
value, and digestibility of the forage. Also im- 
portant to know are the animal requirements 
for food; supplemental feeding alternatives be- 
come important when the food supply is inade- 
quate. Consequently, the range or wildlife 
manager must (1) measure the value of 
plants consumed by range animals and (2) 
measure the animal responses that reflect eco- 
nomic value of the forage. Because of the prob- 
lems that arise in evaluating the range, much 
time in research is devoted to development and 
use of techniques for studying the range for- 
age. 
Research concerning the role of the rumen 
micro-organisms in ruminant animal nutrition 
dates back to the 1800’s and led to the develop- 
ment of the in vitro rumen fermentation tech- 
nique. This technique has revolutionized the 
nutritional evaluation of range forage during 
the last decade. Forage evaluation studies 
based on this digestion technique involve do- 
mestic animals in feedlots and on rangelands, 
and wild ruminants in cages and in their natu- 
ral habitat. The literature concerning various 
phases of in vitro digestion work with domes- 
tic animals in feedlot studies, is voluminous. 
Literature on in vitro studies for rangelands is 
not extensive but has increased in recent years. 
Wildlife studies are limited but will probably 
increase. 
This paper describes the in vitro digestion 
technique, and discusses its usefulness in range 
and wildlife research. Several recent review 
publications are available.” 
PROCEDURE 
The in vitro fermentation technique simu- 
lates under laboratory conditions natural rumi- 
nant digestion. The in vitro system includes 
fermentation of a substrate with rumen mi- 
cro-organisms in a buffered nutrient medium 
under controlled conditions of anaerobiosis, 
temperature, and pH. One of two types of sys- 
tem is employed—open or closed. In the closed 
Range Scientist, Rocky Mt. Forest and Range Exp. 
Sta., USDA Forest Serv., located at Flagstaff, Ariz., in 
cooperation with Northern Ariz. Univ. Central head- 
quarters for the station is maintained at Fort Collins, 
Colo., in cooperation with Colo. State Univ. 
*Bruggemann et al. 1968; Dougherty et al. 1965; 
Hungate 1966; Johnson 1966; Van Dyne 1962. 
system, the substrate is fermented in a closed, 
impermeable container. This container may be 
a test tube, Erlenmeyer flask, or any other ves- 
sel that can be fitted with a suitable closure. 
Transfers of materials should be minimized. 
For instance, Rogers and Whitmore (1966) 
used a vessel with a built-in filter so filtering 
could be accomplished in the same container as 
digestion. 
The open or continuous-flow system differs 
from the closed system because a semipermea- 
ble or open container is used (Adler et al. 1958, 
Huhtanen et al. 1954; Louw et al. 1949); this 
allows addition and removal of metabolities. 
Investigators want simplicity in design; 
therefore, more complicated in vitro systems 
are disappearing. The closed system will be 
emphasized in this paper since it is universally 
used because of its simplicity. 
Substrate 
The amount of substrate or sample to be 
evaluated varies (0.25-1.0 g.) in different labo- 
ratories (Barnes 1967). Within limits, fineness 
of grind does not affect digestibility if the sam- 
ples are ground finely enough to insure good 
sampling of the small weights of herbage used 
(Tilley and Terry 1963). The limits of fineness 
are exceeded when samples are ground very 
fine by ball milling or as large as 2.5 millime- 
ters (Dehority and Johnson 1961; Minson and 
Milford 1967). Fine grinding by ball milling 
was assumed to disrupt the cell walls of the 
plant structure, while the large particles were 
apparently not digested completely. Van Dyne 
(1962) indicates a maximum particle size of 1 
millimeter for in vitro digestion work. No dif- 
ference was found in drying samples at either 
40° C. or 100° C. compared with freeze 
drying. Drying at 100° C. has a marked effect 
only if continued longer than 4 days (Tilley 
and Terry 1963). 
Buffer and Nutrient Media 
The artificial saliva or buffer solution gener- 
ally used (McDougall 1948) contains phos- 
phate-bicarbonate buffers, which are saturated 
with carbon dioxide. The basis for this solution 
has been the chemical analysis of sheep saliva. 
There have been alterations, additions, and 
subtractions for individual laboratory tech- 
niques. For instance, urea, glucose, vitamins, 
or other growth factors are added to the basic 
nutrient medium (Donefer et al. 1960; John- 
son 1966). A relatively large volume of buffer 
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