C. Due to the relative simplicity and repeata- 
bility of the acid detergent lignin method, it 
has been used in most of the more recent re- 
ports citing the lignin ratio technique. 
Early studies (Bath et al. 1956; Lesperance 
et al. 1960a) indicated that salivary contami- 
nation significantly modified the composition of 
fistula forage samples by increasing the ash 
content. Other studies have indicated differ- 
ences in chemical composition of fistula sam- 
ples as compared to hand-clipped or hand-fed 
forages (Connor et al. 1963; Hoehne et al. 
1967). In the studies by Hoehne et al. (1967), 
no change was observed in the acid detergent 
lignin content of squeezed or nonsqueezed eso- 
phageal samples as compared to samples of 
two hand-clipped grass species. However, solu- 
ble carbohydrate fractions were lower in the 
fistula samples. In contrast, Lesperance et al. 
(1960a) and Ridley et al. (1963) reported that 
rumen samples from animals which were evac- 
uated prior to grazing were higher in lignin 
contents than hand-harvested forage samples. 
Connor et al. (1963) reported that the concen- 
tration of lignin was higher in rumen fistula 
forage samples than in various hay and pasture 
feeds. 
McCann et al. (1967)' noted wide differ- 
ences between the acid detergent lignin content 
of available range herbage and rumen fistula 
forage (table 2). Lignin content in these fistula 
samples averaged 68 percent higher than the 
average lignin content in the four major grass 
species available. In every instance, the indica- 
tor content of the fistula forage samples was 
greater than that of any single grass sample 
analyzed. Some of the differences noted be- 
tween the herbage and fistula forage samples 
may be due to selective grazing. However, sim- 
ilar discrepancies in lignin content of rumen 
fistula and hand-fed forage samples have been 
reported by McCann and Theurer (1967) and 
by Lesperance et al. (1967). 
Lesperance and Bohman (1964) reported 
that the addition of water or of artificial saliva 
*See footnote 4, p. 111. 
TABLE 2.—Percent acid detergent lignin in 
forage samples collected by hand clipping and 
rumen fistula >? 
Period 
Sample Aver- 
collection 1 2 3 4 5 age 
Hand clipped ° 5.1 48 44 47 4.7 447 
Rumen fistula*_. 86 76 7.5 82 7.8 7.9 
* See footnote 4, p. 111. 
* Dry-matter basis. 
° Average lignin content in the four major grass 
species available on a ponderosa pine range in northern 
Arizona during the 1967 grazing season. Values for in- 
dividual species varied from 4.0 to 6.1 percent lignin. 
* Average of samples obtained from four steers. 
to hay samples, followed by drying, increased 
acid detergent lignin and decreased nitrogen- 
free extract as compared with the original hay 
samples. Similar results were also observed 
when rumen fistula forage samples were com- 
pared to original hay samples. Their studies 
also indicated that drying temperature had a 
significant influence on lignin and carbohy- 
drate composition of the various samples.The 
lignin content of oven-dried (65° C.) samples 
was significantly greater than that of samples 
vacuum-dried at 25° C. or frozen and lyophil- 
ized. In later studies, Smith et al. (1967) found 
that lignin was significantly higher in oven- 
dried (65° C.) samples of rumen fistula forage 
or feces as compared to freeze-dried samples, 
while nitrogen-free extract was significantly 
lower. Ash content was significantly higher in 
all types of oven-dried samples. Absolute dry 
matter percentage was lower, suggesting the 
possible loss of dry matter during oven drying. 
These studies suggest that the higher lignin 
content of rumen fistula forage as compared to 
original forage may be an artifact largely at- 
tributable to sample preparation. Absorption 
of soluble carbohydrate in the rumen during 
forage collection by the grazing animal may 
also contribute to this problem. 
Lesperance et al. (1967) reported that dry- 
matter digestibility estimated by the lignin-ra- 
tio technique was altered by the drying method 
used for fistula forage and fecal samples. Esti- 
mates of intake and digestible dry matter 
based on oven-dried samples did not differ sig- 
nificantly from actual values, but were signifi- 
cantly overestimated with freeze-dried sam- 
ples. Van Dyne and Lofgreen (1964) reported 
no significant difference in digestion coeffi- 
cients calculated by the lignin ratio when for- 
age and fecal composition were on a dry matter 
basis, silica free or organic matter basis; 
this indicates that ash from salivary con- 
tamination of esophageal fistula forage sam- 
ples had no significant influence on the estima- 
tion of digestion coefficients. The apparent dif- 
ferences in acid detergent lignin content of 
hand-fed versus fistula forage has been pri- 
marily associated with forage obtained from 
rumen rather than esophageal-fistulated ani- 
mals. Further studies are needed to clarify 
chemical changes in fistula samples, as well as 
the effect of sample preparation on the use of 
lignin as an internal indicator for estimating 
digestibility. 
Determining percent recovery of acid deter- 
gent lignin from animals consuming range for- 
age in many instances is not feasible. 
Harvesting of range forage for conventional 
digestion studies is not often possible because 
of the heterogeneous nature or scarcity of the 
herbage. Apparent digestibility of acid deter- 
gent lignin by cattle fed alfalfa hay has been 
determined in studies conducted at the Arizona 
113 
