EVALUATION OF THE TWO-STAGE TECHNIQUE FOR THE IN VITRO

ESTIMATION OF
THE DRY MATTER DIGESTIBILITY OF CORN SILAGE

R. J. BOILA, J. D. ERFLE, and F. D. SAUER

Animal Research Institute, Research Branch, Agriculture Canada, Ottawa, Ontario
KlA 0C6. Contribution no. 869, received I3 JuLy 1979, accepted 3 Nov. 1980.
Borr-n, R. J., Enplr, J. D. nNo Seunn, F. D. 1980. Evaluation of the two-stage
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20

technique for the in vitro estimation of the dry matter digestibility of corn
silage. Can. J. Anim. Sci. 60: 367-378.
The two-stage Tilley and Terry technique (incubation with rumen fluid followed by an
acid-pepsin digest) , used to estimate dry matter (DM) digestibility of forages in vitro,
was evaluated with oven-dried corn silage as a substrate. The effect of weight of
substrate (100-3000 mg), continuous shaking of incubations for the period of
incubation with rumen fluid, number of bacteria present in the inoculum, and the
contribution of bacterjal dry matter to residual feed DM was measured. Percent dry
matter digested decreased linearly as weight of substrate per incubation tube
increased. Continuous shaking, as opposed to intermittent mixing (twice daily) during
incubation with rumen fluid, increased the rate of DM disappearance and resulted in
higher digestibility coefficients. Both the volume of inoculum and the number of
bacteria present in that volume of inoculum influenced the percent DM digested.
For personal use only.

Bacteria contribute weight to residual feed DM unless steps are taken to remove them
by centrifugation or solubilization.

La technique en deux 6tapes Tilley et Terry (incubation avec du liquide ruminal suivie
d'une digestion d la pepsine acide) servant ) estimer la digestibilit6 de la matibre sbche
des fourrages in vitro a 6t6 6,valu6e au moyen d'ensilage de mais s6ch6 au four comme
substrat. On a ainsi mesur6 I'effet du poids du substrat (100 h 3000 mg), de I'agitation
continue des incubats pendant la p6riode d'incubation avec le liquide ruminal, le
nombre de bact6ries de I'inoculum et la contribution de la matidre sdche bact6rienne d
la teneur en matibre sdche r6siduelle de I'aliment. Le pourcentage de matibre sbche
dig6r6e d6croit de fagon inversement proportionnelle b I'augmentation du poids du
substrat par tube d'incubation. L'agitation continuelle, contrairement d intermittente
(deux fois parjour) au cours de I'incubation avec la liquide ruminal, accroit le taux de
consommation de la matibre sbche et am6liore les coefficients de digestibilit6. Le
volume de I'inoculum et le nombre de bact6ries qu'il contient influent sur la digestion
de la matibre sbche. Les bact6ries contribuent I accroitre la matibre sbche 16siduelle de
I'aliment si l'on ne prend pas les mesures n6cessaires pour les enlever par
centrifugation ou solubilisation.

A two-stage in vitro technique was de- Several factors influence the final in vitro
veloped by Tilley and Terry (1963) for the estimate of DM digestibility. The largest
estimation of the dry matter (DM) digestibil- single source of variability is the inoculum
ity of forages. The method has been widely (Johnson 1969), which is influenced by the
used for this purpose and has been type of diet fed to the donor animal (Knipfel
recommended by Tilley et al. (1960) and and Troelsen 1966). Additional variation is
TilleyandTerry(1963)fortheclassification due to the particle size of the substrate,
of groups of forage samples relative to one weight of substrate, ratio of volume of
another in terms of their DM digestibilities. inoculum to buffer (Mcleod and Minson
Can. J. Anim. Sci. 50: 367-378 (June 1980)

36'/
 CANADIAN JOURNAL
I 969), and length of time for the first stage of
the technique (Troelsen and Hanel 1966).
The Tilley and Terry (1963) method in its
original form is still widely used and is one
of the best methods for the prediction of in
vivo dry matter digestibility (apparent
digestibility) for forages (Scales et al. 1974:
Aerls et al. 1911; Barnes and Marten 1979).
Numerous modifications to the original
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20
method have been tested in attempts to
reduce the variability from the factors listed
above (Alexander and McGowan 1966; Van
Soest et al. 1966; Goering and Van Soest
1970; Nelson et al. l9l2; Sayre and Van
Soest | 972; Grant et al. 1974).
Many laboratories use the Tilley and
Terry procedure but introduce modifications
to suit their own needs and laboratory
situations, perhaps not fully realizing the
consequences of these changes. The objec-
tives of this study were to examine the Tilley
and Terry procedure with oven-dried corn
For personal use only.
silage as a substrate with regard to the
relationship between in vitro DM disappear-
ance and ( 1 ) recovery of individual and total
volatile fatty acids and weight of substrate
per incubation, (2) the effect of continuous
mixing during incubation with rumen fluid,
(3) the volume of inoculum and the bacterial
counts in that inoculum, and (4) the
contribution of bacterial DM to residual feed
dry matter used in the calculation of percent
DM digested.
MATERIALS AND METHODS
General Incubation Technique
Dry matter digestibility was determined in vitro
using a modification of the two-stage technique
of Tilley and Terry (1963). Rumen fluid was
obtained from a fistulated cow approximately 3 h
after feeding. The cow was fed either a diet
containing 4OVa corn silage,407c alfalfa hay and
2OVo haylage (alfalfa-grass mixture) on a DM
basis (diet A) or a diet of badly wearhered poor
quality alfalfa hay (diet B). Rumen fluid from the
cow when fed diet A was used in all studies
reported in this paper; only when the relationship
between percent DM digested and bacterial
counts per incubation was examined was
inoculum used from the same cow but fed diet B.
OF ANIMAL SCIENCE
Corn silage (33.07o dry matter, 7.387c crtde
protein on a DM basis) was oven-dried at 100'C
for 24 h. The oven-dried corn silage was ground
in a laboratory mill fitted with a l-mm screen.
Weighed quantites of dried corn silage, in
triplicate, were mixed with a modified
McDougall's buffer (McDougall 1948). In the
modified McDougal['s buffer, MgSO1.7H2O
(0.12 glL) was substituted for MgCl2 (Johnson
1966). Once the salts were dissolved, the buffer
was gassed for 20 min with CO, with a dispersion
tube, after which the buffer was adjusted to pH
6.9. In preliminary investigations it was found
that the in vitro DM digestibility of oven-dried
corn silage was not improved by the inclusion of
NHaCI or (NH4)rSO1 in the buffer. The corn
silage and 40 mL McDougail's buffer were
preincubated in 80-mL glass incubation tubes
(200 x 25 mm) at 39'C in a water bath for
approximately I h. Ten milliliters of rumen fluid,
after being strained through four layers of
cheesecloth (Fisher Scientific Company, Fair
Lawn. N.J., U.S.A.), were added to incubation
tubes. Immediately after the 10 mL of inoculum
was added, the head-speace of each incubation
tube was flushed with COz, and each incubation
tube was sealed with a Bunsen gas release valve,
and incubated anaerobically for 48 h in a water
bath at 39'C. Incubation tubes were mixed
manually with a swirling action after 3, 6,24 and
30 h of incubation for the first stage of the
procedure. Unless otherwise indicated, this
procedure was used throughout.
After the rumen fluid digest, the contents of
each incubation tube were acidified directly with
4 x I mL of 3 N HCI containing a total of 0. I g
pepsin ( I : 10 000; Nutritional Biochemicals
Corporation, Cleveland, Ohio, U.S.A.). All feed
particles were washed from the sides of the
incubation tubes and from the stoppers. The
acid-pepsin digest was incubated aerobically for
48 h in a water bath at 39'C. Undigested DM was
recovered by filtering the acid-pepsin digest
through tared glass microfibre filters (Whatman
GF/A). Filters plus residual DM were dried at
55'C under vacuum for a minimum of48 h, and
weighed to determine the quantity of residual DM
recovereo.
A minimum of four incubations of rumen fluid
plus buffer to a total volume of 50 mL were
prepared as blank incubations. Average DM
recovered from the blank incubations was
substracted from the residual dry matter before an
in vitro estimate of dry matter digestibility
 BOILA ET AL. DIGESTIBILITY OF CORN SILAGE
-
(DMD) was calculated according to the fbllowing
equatlon:
DMD:[Substrate DM - (Residual DM - Blank
DM)l (100)/(Substrate DM)
Volatile Fatty Acid Analysis
Filtered incubation media, with added intemal
standard (isocaproic acid), were injected directly
onto a glass column (6 m long, 3 mm i.d.)
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containing l5Vo SP-1220 and 1% H3POa on
Chromosorb W AW (100-120 mesh) (Supelco
Inc., Bellefonte, Penn., U.S.A.), fitted into a
Hewlett Packard 58404 gas chromatograph. The
oven temperature was programmed at a rate of
1.5'C/min from 135 to 175"C. The flow rate of
nrtrogen as a carrier gas was 35 ml/min.
Millimoles of acetic, propionic, isobutyric,
butyric, isovaleric, valeric and caproic acids, and
of total volatile fatty acids (VFA) recovered,
were calculated separately for each incubation.
Bacterial Counts
Bacterial counts were obtained using the
For personal use only.
procedure described by Hungate et al. (1971).
Rumen fluid, incubation media or filtrates of
incubation media were homogenized for 2 min
(90 volt setting on a rheostat) with an
Omni-Mixer (Ivan Sorvall Inc., Norwalk, Conn.
U.S.A.) before being diluted l:1 (vol/vol) with
8% formaldehyde. Appropriate dilutions were
made with 87o formaldehyde. Bacteria were
stained with crystal violet and counted in a
Thoma counting chamber (Hawksley, England)
under oil immersion at a masnification of 1000
under phase contrast.
The Effect of Weight of Substrate on Dry
Matter Digested and Volatile Fatty Acid
Production
Total dry matter (residual DM minus blank DM)
digested per incubation, percent DM digested,
and individual and total volatile fatty acid (VFA)
recovered from each incubation were determined
forpreparations containing substrate at 100-3000
mg per incubation. The pH of samples was
measured after 48 h of incubation with rumen
fluid. The volume of incubation medium was
determined by weighing incubation tubes with
and without the incubation medium present;
allowance was made for the weight of residual
DM present in each incubation. The millimoles of
individual VFA recovered at each weight of
substrate (minus millimoles present in a blank
369
incubation) were calculated as a glucose
equivalent; this estimate of DM disappearance
was compared to the DM residue (residual DM
minus blank DM) obtained after 96 h of
digestion. Nitrogen, crude fat and ash present in
the original weight of substrate were substracted
from the DM digested before this comparison was
made. The stoichiometry between VFA reco-
vered and DM metabolized by rumen bacteria
was calculated with the assumption that I mole of
glucose (molecular weight of 162 for glucose as
polyglucose) was equivalent to 2 moles of acetic
or propionic acids , or to 1 mole of butyric , valeric
or caproic acids (Stadtman et al. 1949; Ladd
19591 Czerkawski 1978).
The Effect of Continuous Shaking of
Incubations
Incubations containing 500 mg of substrate were
rerminated after 12, 24 , 36, 48 , 72 and 96 h for
the first stage of the procedure; the rumen fluid
digest was immediately followed with a 48-h
acid-pepsin digest. Blank incubations were
terminated at0, 12 and48 h; DM obtained at48 h
in the blank preparations was used to calculate
residual feed DM at all times except 0 and 12 h.
This arrangement of incubations was duplicated;
one set of incubations was mixed intermittently
by hand aI 3 , 6, 24 , 30, 49 and 72 h for the first
stage of the procedure; the second set of
incubations was shaken continuously (55 oscilla-
tions/min) in an upright position, in addition to
being mixed by hand at the times specified for the
first set of incubations during the rumen fluid
digest. The same rumen fluid was used for both
sets of incubations.
The Effect of Bacterial Numbers and Volume
of Inoculum
Inoculum was obtained from a fistulated cow fed
either diet A or diet B; each source of inoculum
was prepared separately. Inoculum volumes of
1.0. 2.5. 5.0. 10.0. 15.0 and 20.0 mL were
incubated with 250, 500, 1000 and 1500 mg of
substrate. The total volume of inoculum plus
buffer was 50 mL. Bacterial counts per
incubation were obtained by multiplying the
counts present in the original rumen fluid by the
volume of rumen fluid included in each
incubation.
The Contribution ofBacteria to Residual Feed
Dry Matter
Duplicate incubation tubes were prepared with 0,
 310 CANADIAN JOURNAL
100, 250, 500, 1000 and 1500 mg substrate. A
lO-mI- aliquot from each tube was taken after
48 h of incubation, homogenized and prepared
for bacterial counts as described above. The
remaining 40 mL was filtered through Whatman
GF/A glass fibre filters, and the filtrate
homogenized and prepared for bacterial counts.
Incubations (in triplicate) with substrate
weights of
100, 250, 500, 1000 and 1500 mg
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20
were prepared to determine if percent DM
digested was comparable to that obtained in other
trials.
Statistical Analyses
Linear regression analyses were undertaken with
percent DM digested, total VFA recovered per
incubation or DM disappearance per incubation
as thedependent variable, and weight ofsubstrate
per incubation as the independent variable.
For total VFA recovered, individual VFA
recovered and total DM disappearance, standard
errors were estimated from the error mean square
of a one-way analysis of variance. One-way
For personal use only.
analysis of variance was used to compare percent
DM digested within and between incubations
mixed intermittently or shaken continuously,
each time the first-stage rumen fluid digest was
terminated.
At each weight of substrate (250, 500, 1000
and 1500 mg) percent DM digested for both
sources of inoculum, at all volumes of inoculum,
were combined for a polynomial regression
analysis (degree of polynomial equal to 3). For
this analysis, percent DM digested was the
dependent variable and volume of inoculum per
incubation or bacterial counts Der incubation was
the independent variable.
Linear regression analysis, one-way analysis
of variance, and polynomial regression analysis
procedures were those described by Snedecor and
Cochran (1967).
RESULTS AND DISCUSSION
The Effect of Weight of Substrate on DM
Digested and VFA Production
Dry matter digestibility coefficients ob-
tained with the two-stage technique are
determined with measurements of DM
disappearance. Factors that influence DM
disappearance include bacterial degradation
of substrate, enzymatic digestion of sub-
strate in acid-pepsin, solubility of substrate
OF ANIMAL SCIENCE
in buffer and acid, and the retention of
bacterial mass in the filtered residue.
Mcleod and Minson (1969) observed that
DM digestibility decreased with increasing
sample size. In agreement with this, the
present results show that the percent DM,
digested decreased linearly when the amount
of substrate per incubation was increased
(Fie. 1).
Total VFA production (r2 : 0.992) and
DM disappearance (r'z : 0.998) increased
linearly with weight of substrate per
incubation up to a level of 1250 mg substrate
(Fig. 2). Between substrate levels of 2000
and 3000 mg, DM disappearance increased,
but VFA production did not (Fig. 2). This
suggests that at these levels the substrate
may be solubilized but not completely
metabolized by the rumen bacteria. Evi-
dence for this is also found in Table l, which
suggests that the percent DM disappearance
accounted for as VFA was reduced at the
highest level of substrate. The same pattern
was observed for individual VFA (Fig. 3)
with the exception of butyrate and valerate
whose production continued to increase at
the higher substrate levels. A possible
explanation for this may be the observation
that the pH of these incubations decreased
with increasing substrate levels, and that this
caused a change in the microbial population.
The pH of McDougall' s buffer was 6. 9; after
48 h of incubation with rumen fluid, the pH
of incubation media dropped to 6.5 with 750
mg of substrate, and to 5.5 with 3000 mg of
substrate.
In Table 1, corrected DM disappearance is
related to the production of glucose
equivalents which were calculated from
VFA production as described under Mate-
rials and Methods. With this calculation it is
possible to distinguish that portion of DM
disappearance due to solubility of substrate,
and that due to metabolism of substrate to
VFA by bacteria. Good stoichiometry was
obtained with substrate levels up to 1250
mg. At higher substrate levels the appear-
ance of glucose equivalents decreased
markedlv. This mav have been due to the
 BOILA ET AL. DIGESTIBILITY OF CORN SILAGE 371
-

u
F
o
U

d
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u
F
F

o a
F
zu
o Y= 73.5-0.0097X
(r2=O.97: Sy.x= 1.5)
U

0 1000 2000
For personal use only.

3000
WEIGHT OF SUBSTRATE (mg)

Fig. I . The effect of weight of substrate dry matter present per incubation upon percent dry matter
digested, after completion of incubation with rumen fluid and an acid-pepsin digest. Each point
reDresents a mean of three values.

Table l. Calculation of individual volatile fatty acids recovered from incubations as a glucose equivalent

Dry Corrected VFA as Glucose equivalent

Wt of matter dry matter glucose as a percent of
substrate disappearance disappearance equivalent corrected dry matter
(mg)T (mg)l (mg)$ (mg)// disappearance

102.5 7 5.',] 67.4 65 96.4

25r.1 180.7 160.4 r38 86.7
503. 1 347.O 306.3 7'7.4
'7 51.9 494.6 433 .8 iJl 16.3
1004.2 639.0 55'7.7 423 75.8
1252.9 502 '74.5
7',7 s.1 613.1
1503 .2 863 .5 '7
4t .9 518 69.8
2002.9 l039.6 8'77.5 633 '72.1
3005. l 1396.9 I t53 .7 758 65.7
tAir-dry basis.
ln : 3 at each weight of substrate.
$Corrected dry matter disappearance was obtained by substrating the milligrams ofnitrogen, crude fat and ash present
inrheweightofsubstrateincludedineachincubation(7cN: l.14,Vocrudefat:2.1l,Voash:4.85intheoven-dried
corn silage used in this experiment).
//For an explanation of glucose equivalent see Materials and Methods.
 3'72 CANADIAN JOURNAL OF ANMAL SCIENCE

z 8.0

F z
3 z.o TOTAL MILLIIVOLES VFA RECOVERED 400 k
PER INCUBATION (SE = 0.16) o
l
= 200 z
-- 6.0
c(
o u
u
ooo ff
F
o
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U
H +.0 800 :

;r TOTAL ms DRY IVIATTER DIGESTED 600 k

U
F
3.0
PER INCUBATION (SE= 9.0)
q)
U
CE
^AA
E

>
= 1.0 200 a
F
a
F

0 1000 2000 3000

WEIGHT OF SUBSTRATE (mg)

For personal use only.

Ftg. 2 .
The effect of weight of sub strate present per incub ation upon the total of volatile fatty acid s
recovered from each incubation and dry matter disappearance per incubation. Standard errors (SE)
were calculated using error mean square from a one-way analysis of variance of the data. Each point
represents a mean of three values.

partial digestion of substrate by bacteria to percent DM digested was not significantly

soluble intermediates (i.e., cellobiose,
glucose, lactic acid) without formation of
VFA. Based on the stoichiometry of VFA
production, these results suggest that
corrected dry matter disappearance is a valid
measure of digestibility of substrate levels of
500 to 1000 mg.
The Effect of Continuous Shaking of
Incubations
An estimate of the maximum percent DM
digested of oven-dried corn silage (75. l7o)
was obtained when incubation tubes were
shaken continuously (Table 2). This digesti-
bility value was reached at 48 h with
continuous shaking (no significant differ-
ence (P > 0.05) between 48 and 96 h, Table
2) but not at 48 h with intermittent mixing
(significant difference (P < 0.01) between
48 and 96 h,
Table 2). After 96 h of
incubation of the rumen fluid disest. the
different (P > 0.05) for incubations mixed
intermittently and shaken continuously
(Table 2). Tilley and Terry (1963) have
standardized the first stage of the technique
as 48 h in duration. Continuous shaking of
incubations increased the rate ofdigestion of
the 500 mg of substrate to the extent that the
maximum percent DM digested was esti-
mated when the rumen fluid digest was
incubated for 48 h. With intermittent
mixing, as recommended by Tilley and
Terry ( I 963) , the digestibility of oven-dried
corn silage would have been underestimated
by four percentage points (Table 2).
The substrate in incubation tubes kept in a
vertical position forms a dense mat at the
surface of the incubation medium; this was
particularly evident when weights of sub-
strate were greater than 500 mg. Intermittent
mixing (4 times daily), as described by
Tilley and Terry (1963), permits formation
 BOILA ET AL. DIGESTIBILITY OF CORN SILAGE -)t-)
-

z 2.O
tr
c0
1.0

=
q
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0
U
U
0.6

u
E.

; 0.4

o.2
I
u
J
0

o
L 0.06
o
For personal use only.

o
U
0.04
E
f
E 0.02

WEIGHT OF SUBSTRATE (mg)

Fig. 3. The effect of weight of substrate present per incubatron upon the recovery of individual
volatile fatty acids from each incubation. A. acetic (.-., SE: 0.083), propionic (o-o' SE:
0.063), butyric (o-o, SE : 0.029). B. valeric (o-o, SE : 0.012), caproic (o-o, SE : 0.048). C.
isovaleric (.-., SE:0.0014), isobutyric (o-o, SE: 0.0019). Standard errors (SE) were
calculated using error mean square from a one-way analysis of variance. Each point represents a mean
of three values.

of this mat of substrate. Since some and Sayre and Van Soest (1972). These
accumulation of substrate at the surface of authors observed that good DM digestibility
the incubation medium was also observed values could be obtained with only occa-
when incubation tubes were shaken continu- sional agitation when either screwcapped
ously, it was necessary to mix these tubes by tubes or 125-mL Erlenmeyer flasks were
hand. Mellenberger et al. (1970) found that used. From this it may be concluded that
with slow rotation of sealed incubation tubes continuous mixing of incubation tubes with
in a horizontal position, there was better Bunsen values, incubation in screwcapped
contact between substrate and medium with tubes in a horizontal position, or incubations
less mat formation. in 125-mL glass Erlenmeyer flasks will
A different approach to particle dispersion increase DM digestibility above that ob-
was used by Goering and Van Soest (1970) rained with the original Tilley and Teny
 J t+ CANADIAN JOTIRNAL OF ANIMAL SCIENCE

Table 2. Effect of intermittent mixing and continuous Sy (Table 3) were found with 500 mg of
shaking of incubation tubes upon percent dry matter
substrate (Fig. a). A higherr2 and a lowerSj
disested in vitro
indicate that bacterial counts would account
Time lntermittent Continuous for a greater proportion of the variation in
(h) mixing shaking SE percent DM digested than would the volume
0 25.5 A of inoculum. At 1500 mg of substrate,
t2 39.6 Ba 43.5 Bb 1.17* factors other than inoculum volume or
.A 49.6 Ca 56.8 Cb 1.05+* bacterial counts per incubation might have
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36 60.5 Da 67.4Db 1.05**

48 68.3 Ea 12.2 Eb 0.94.*
72 13.4 Fa 14.8 Eb 0.35*
96 74.8 F 15.1 E 0.78 U
SE 0.92** 0.83 ** o
&
lThe zero h percent dry matter digested for intermittent o a
mixing and continuous shaking was one set of three E o
incubations (SE : 1.09) prepared and incubated for 48 h
U
F a
o
F
in acid-pepsin after l0 mL of rumen fluid were added to
each incubation tube. =
,4-F Means within columns without a common letter are
significantly different. F
a,b Means within a row without a common letter are z
U
signifi cantly different. E
U
Significantly different: * : P ( 0.05, 8+ : P < 0.01.
'-l rttl
For personal use only.

0 48't216 20
procedure. The eventual choice of method VOLUME OF INOCULUM (mll PER
INCUBATION
may well depend on the facilities available or
the preference of the investigator.
U
U
The Effect of Bacterial Numbers and
Volume of lnoculum 6
When the fistulated cow fed diet A was U
F
F
a
o
changed to diet B, the number of bacterial
cells decreased from 2.38 x 1010 to 1 .44 x /
E
1010 per mL of rumen fluid. Percent DM
digested in in vitro experiments at each zUF
weight of substrate was combined for a 4
polynomial regression analysis. When per- c
40
cent DM digested (dependent variable) was ttttll
01020304050
regressed upon bacterial counts per incuba- BACTERIAL coUNTs (Xlo-10) PER
tion, as opposed to volume of inoculum, as INCUBATION

the independent variable, the proportion of
the variation in the dependent variable
attributed to its regression on the indepen-
dent variable (r2) was higher at weights of
substrate of 250,500 and 1000 mg, but not at
1500 mg (Table 3). Standard errors for the
estimate of the dependent variable (Sy) were
lower when bacterial counts per incubation
was the independent variable at weights of
250, 500 and 1000 mg, but not at 1500 mg
(Table 3). The largest differences for 12 and
Fig. 4. The relationship between percent dry
matter digested (I) as the dependent variable and
volume of inoculum per incubation (A, Y : 47 .7
+ 5.30x -0.41-r2 + 0.01 xs;12 :0.6a; Sy :
4.75) or bacterial counts per incubation (B,Y :
47.5 + 2.55x -0.08912 * 0.00096x3;r2 - 0.'79:
Si - 3.61) as the independent variable for500 mg
as a weight of substrate per incubation. Inoculum
was obtained from a fistulated cow fed corn
silage: alfalfa hay: haylage (closed circles) or a
poor quality hay (open circles). Each point
represents a mean of three values.
 BOILA ET AL. DIGESTIBILITY OF CORN SILAGE 3'75
-
Table 3 . The 12 values and the standard errors of the estimate (Sj) from a polynominal regression analysis of degree
equal to three for the relationship between percent dry matter digested and volume of inoculum or bacterial counts p€r
rncuDanon.

Weight of substrate per incubation (mg)

Independent variable 250 1000 1500

r'l Volume of inoculum o.73 0.64 0.84 o.72

Bacterial counts 0.84 0.'79 0.93 0.70
sif Volume of inoculum 4.39 / 1< 1.95 1.33
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Bacterial counts 3.60 3.61 I.JJ 1.36

ir2, the proportion of the variance ofthe dependent variable that can be attributed to its regression on the independent
variable.
tSy, standard error of the estimate of the dependent variable.

limited percent DM digested. It was
concluded that percent DM digested de-
pended to some degree upon the number of
bacteria added to each incubation. Mcleod
and Minson (1969) did not do bacterial
counts but noted that there was an interaction
between percent DM digested and volume of
inoculum.
For personal use only.
were isolated from rumen fluid from the cow
when fed diet A, using the differential
centrifugation procedure of Meyer et al.
(196'7). The relationship of bacterial DM to
bacterial counts was 1.75 mg DM per 1010
bacteria. With 50O mg of substrate (Table 4),
3.43 x 10e bacteria per mL of incubation
medium were retained with the residual DM

Total bacterial counts per incubation do after the rumen fluid digest; bacteria from 50
not completely account for the differences in mL of incubation medium could contribute
DM digestibility (Figs. 4A and 48) because 30 mg DM to residual feed DM (1.75 x 10-to
when bacterial counts were equalized (Fig. mg per bacterium x 3.43 x lOe bacteria per
48). some differences due to the source of mL x 50 mL) after 48 h of incubation.
inoculum still persisted. These differences
could be due to changes in the rumen Table 4. Bacterial counts present in the original
microflora (Slyter and Weaver 1972), a incubation medium and recovered in the filtrate after a
requirement for added glucose or nitrogen in 48-h incubation with rumen fluid and percent dry matter
rumen fluid from cows fed a poor quality diet digested after the complete procedure
(Nelson et al. 1972), or the need for
(x 10s)
Bacterial counts
unspecified components that may be lacking at 48 hl
in the rumen fluid of such cows (Hungate
Wt of Original 7o dry
1960; Bryant 1973; Bales et al. 1916). matter digested
subskate incubation
f mo) medium Filtrate at 96 hl

The Contribution of Bacteria to Residual 0 1.08$ 1.06

Feed Dry Matter 100 3.09 r.28 / t.6
4.00 71.0
The results in Table 4 indicate that the 250
4.16
1.34
0.'73 69.0
500
number of bacteria present after 48 h 1000 4.'11 o.21 &.8
increased in response to an increase in 1500 '7.38 1.97 58.2
weight of substrate and the number of fFrom a volume of 50 mL, 10 mL was prepared and
bacteria retained with the residual DM counted directly, while 40 mL was filtered and the filtrate
increased as weight of substrate increased. was prepared for counting. Only one incubation at each
Bacteria may contribute to the weight o[ weight of substrate was counted.
f Each measure of percent dry matter digested is a mean
residual DM recovered. of three values.
In order to estimate the extent that bacteria $The rumen fluid used in this study contained 1.23 x
may contribute to residual feed DM, bacteria 1010 bacteria oer mL.
 376 CANADIAN JOURNAL
Based on the composition of rumen bacteria
(Hoogenraad and Hird 1970) and the
digestibility of bacterial protein in pepsin
(Bergen et al. 1968), the digestibility of the
DM of rumen bacteria in acid-pepsin was
estimated at 50Vo. In the residual DM
recovered from 500 mg of substrate,
undigested bacteria could contribute l5 mg
of DM, or account for a difference of
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20
approximately 3 percentage points in percent
DM digested.
Centrifugation of incubation tubes after
48 h of incubation was a part of the original
technique developed by Tilley and Terry
(1963). After a low speed centrifugation step
(18009 for 15 min), the supernatant, which
contained most of the bacteria from the
incubation medium, was discarded. The
precipitate, which contained only those
bacteria associated with feed particles, was
resuspended in acid-pepsin and digested
enzymatically. Since this centrifugation step
For personal use only.
was time-consuming, and some forages did
not sediment readily, the centrifugation step
was replaced with a step where incubation
media were acidified with pepsin in an HCI
solution (3 N HCI was used in this study).
Residual DM was recovered when the
incubation medium was filtered 48 h after
direct acidification (total of 96 h for the
procedure). With direct acidification at
48 h, DM of bacterial origin is recovered
with residual feed DM when filtration is
undertaken after 96 h (Table 4). This
bacterial mass can represent a difference of
three percentage points in the in vitro
estimate of DM digestibility.
Tilley and Terry (1963) found no
consistent decrease in percent DM digested
when direct acidification was included as a
part of their procedure. However, in
agreement with out study, Schmid et al.
(1975) found the percent DM digested to be
reduced 2-3 percentase points for corn and
sorghum silages when direct acidification
was used after 48 h. Direct acidification of
incubation media, after the fermentation
step, has been used (Troelsen and Hanel
1966; O'Shea etal. 1972: Scales et al.1974
OF ANIMAL SCIENCE
Tinnimit and Thomas 1976) and is particu-
larly advantageous when an attempt is made
to automate the technique (Alexander and
McGowan 1966). When subsequent
between-laboratory comparisons are made,
it is important to recognize the potential error
introduced by the replacement of the
centrifugation step with direct acidification.
A modification of the Tilley and Terry
(1963) procedure has been reported by Van
Soest et al. (1966) and Goering and Van
Soest (1970). Centrifugation and acidifica-
tion of incubations are replaced by an
analysis for neutral detergent fibre wherein
bacterial cells are solubilized and not
recovered with the filtered residue. In vitro
estimates of percent DM digested obtained
with the Van Soest modification are higher
than the estimates obtained with the Tilley
and Terry procedure. This difference,
attributed by Van Soest et al. (1966) to the
recovery of bacterial DM with residual feed
DM in the Tilley and Terry procedure, is
several-fold larger (Aerts et al. 1977) than
the 3 percentage points calculated for the
data at 500 mg of substrate in Table 4.
Further studies are needed to establish the
relationship between the Van Soest modifi-
cation and the original Tilley and Terry
procedure with respect to the composition of
DM disappearance and residual DM.
SUMMARY
The implications of several modifications to
the two-stage technique presented by Tilley
and Terry (1963) have been studied with
oven-dried corn silage as a substrate.
With 500 + 5 mg of substrate, there was
less variation among triplicate analyses than
with 100 or 250 mg of substrate, and the
buffering capacity of the incubation medium
was not exceeded.
When centrifuge tubes or other narrow
incubation vessels are used, continuous
mixing of incubations is preferable to
intermittent mixing. With continuous mix-
ing, the variance for percent DM digested
was smaller, and a higher DM digestibility
 BOILA ET AL.
-
coefficient was measured with 48 h for the
period of incubation with rumen fluid.
Both the volume of inoculum and the
number of bacteria present in that volume of
inoculum contributed to the variability
present in the two-stage technique. A small
amount of variability may be due to
components present in the rumen fluid.
The contribution of bacterial mass to
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20
DIGESTIBILITY OF CORN SILAGE 3'77
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Serv., U.S. Dep. Agric. Agric. Handb.379.
GRANT, R. J., VAN SOEST, P. J. and
McDOWELL, R. E. 1974. Influence of rumen
fluid source and fermentation time on in vitro true
dry matter digestibility. J. Dairy Sci.57:
1201-1205.
HOOGENRAAD, N. J. and HIRD, F. J. R.
1970. The chemical composition of rumen

bacteria and cell walls from rumen bacteria. Br.

residual feed DM can be reduced or J. Nutr. 24: 119-127.
eliminated. Centrifugation of tubes after HUNGATE, R. E. 1960. Microbial ecology of
incubation with rumen fluid as recom- the rumen. Bact. Rev. 24:353-364.
mended by Tilley and Terry (1973) removes HUNGATE, R. E., REICHL, J. and PRINS, R.
197 1. Parameters of rumen fermentation in a
free bacteria. Alternately, a neutral deter-
gent extraction procedure has been recom- continuously fed sheep: Evidence of microbial
rumenation pool. Appl. Microbiol. 22: 1104-
mended to eliminate bacterial DM.
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JOHNSON, R. R. 1966. Techniques and
ACKNOWLEDGMENT procedures for in vitro and in vivo rumen studies.
Assistance in the form of a Govemment of J. Anim. Sci. 25: 855-875.
Canada Visiting Fellowship to R. J. Boila is JOHNSON, R. R. 1969. The deveiopment and
gratefully acknowledged. application of in vitro rumen fermentation
methods for forage evaluation. Proc. of the
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AERTS, J. V., DE BRABANDER, D. L., National Conference on Forage Quality Evalua-

COTTYN, B. G. and BUYSSE, F. X. 1977. tion and Utilization (U.S.A.), MI-M18, U.S.
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the organic matter digestibility of forages. Anim.
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