NUTRITION, FEEDING, AND CALVES
In Vitro Methods for Measuring the Dry Matter Digestibility
of Ruminant Feedstuffs: Comparison of Methods
and Inoculum Source
ABSTRACT
We conducted this study to evaluate the new in vitro
system, DAISYII, to determine dry matter (DM) digest-
ibility in ruminant feedstuffs. Results from the DAISYII
were compared to those obtained by the traditional Tilly
and Terry method. The traditional method buffer was
used for both methods. We also compared two sources of
rumen inoculum from sheep and dairy cows. Seventeen
different feeds were tested, grouped into roughage, con-
centrate, and CP supplements. The experiment was
replicated on two different occasions for all feeds and
the two sources of inoculum. The source of inoculum
and the time at which it was collected had no effect on
the in vitro DM digestibility of the feedstuffs in either
of the methods. The DAISYII DM digestibility value
compared well with the traditional method values for
the roughage group; however, for some feedstuffs in the
concentrate and CP supplement groups, the DAISYII
values were significantly higher than the traditional
method values. Regression analysis of the feeds that
resulted in similar values with the two methods re-
vealed that the DAISYII method can be used to predict
in vitro digestibility with relatively small variation.
(Key words: in vitro, dry matter digestibility, inocu-
lum, rumen)
Abbreviation key: IVDMD = in vitro dry matter di-
gestibility, TT = Tilly and Terry.
INTRODUCTION
The in vitro dry matter digestibility (IVDMD)
method has been extensively used to evaluate the nutri-
tional value of ruminant feeds. Since its introduction,
the Tilly and Terry (TT; 1963) IVDMD method has been
largely used to analyze feedstuffs and has been the most
Received December 28, 1999.
Accepted April 20, 2000.
Corresponding author: S. J. Mabjeesh; e-mail: mabjeesh@
agri.hugi.ac.il.
2000 J Dairy Sci 83:2289–2294 2289
S. J. Mabjeesh, M. Cohen, and A. Arieli
Department of Animal Sciences,
The Faculty of Agricultural, Food,
and Environmental Quality Sciences,
P.O. Box 12, Rehovot 76100, Israel
accurate and practical laboratory method available for
predicting digestibility data for ruminants (Goldman
et al., 1987; Stern et al., 1997). The method has been
modified and adapted for starch feedstuff analysis (Au-
frere and Michalet-Doreau, 1988), and various workers
have improved its accuracy of prediction. Different dilu-
tion buffers for the rumen liquor have been developed
to adjust the pH of the inoculum (Grant and Mertens,
1992). Many laboratories have proven the method to
be simple, highly repeatable, and reproducible (Tilly
and Terry, 1963; Goldman et al., 1987; Aufrere and
Michalet-Doreau, 1988; Stern et al., 1997). However,
despite its accuracy at predicting DMD, the method’s
two-stage procedure is time- and labor-consuming, and
each feedstuff has to be incubated separately.
The search for better labor efficiency has led to the
development of the DAISYII apparatus (ANKOM Tech-
nology Corp., Fairport, NY), which allows simultaneous
incubation of different feedstuffs in sealed polyester
bags in the same incubation vessel. With this method,
the material that disappears from the bag during the
incubation is considered digestible. Recently, Holden
(1999) compared the TT and DAISYII methods for pre-
dicting DMD with the buffer recommended by ANKOM
for both systems. Results from that experiment showed
good agreement between the two systems, proving that
DAISYII could be used to predict the IVDMD of forages
and grains. The present study was conducted to deter-
mine the reliability of the DAISYII system for predicting
the IVDMD of different feedstuffs commonly used in
dairy rations, with different inoculum sources (dairy
cows and sheep) and the buffer used in the TT method.
The IVDMD values obtained with the TT method for
the same feedstuffs were used as a reference for com-
parison.
MATERIALS AND METHODS
Experimental Protocol
Three groups of feedstuffs used regularly in dairy
rations on Israeli dairy farms were selected for this
2290 MABJEESH ET AL.

Table 1. Chemical composition of the feedstuffs on a DM basis. For both methods, the incubation inoculum was pre-
% of DM pared by diluting the digesta inoculum with the buffer
Feedstuff OM CP NDF
(Tilly and Terry, 1963) in a 1:4 (vol/vol) ratio and stir-
ring in a water bath at 39°C with purging CO2 until
Forages its use (10 to 15 min later). In the TT trial, 250 mg of
Grass hay 89.5 11.7 58.1
Alfalfa cubes 84.9 16.7 42.9 each sample was placed into 50-ml sterile plastic tubes
Alfalfa hay 88.4 21.8 50.0 (Miniplast Ein-Shemer, 37845, Israel), and 20 ml of the
Clover hay 89.9 10.8 51.0 incubation inoculum was added. The tube was stop-
Concentrates
Corn grain 94.0 9.5 11.5 pered with a Bunsen valve and incubated for 48 h at
Barley grain 96.2 11.3 19.0 39°C. Tubes were gently swirled by hand four times
Wheat grain 96.2 13.5 14.0 every 12 h. For the DAISYII incubation, 500 mg of each
Sorghum grain 97.4 8.8 11.0
Concentrate mix1 93.5 18.2 27.6 sample was inserted into each bag (F57; 5 × 5.5 cm2,
Wheat bran 94.1 15.5 47.5 ANKOM Technology Corporation), which was then
CP supplements heat-sealed and incubated in a digestion vessel (25 bags
Soybean meal 93.0 49.3 14.2
Sunflower meal 91.2 34.5 26.3 per vessel) for 48 h at 39°C. For both methods, each
Corn gluten feed 91.1 29.9 39.0 sample was incubated in six replicates for each source
Cottonseed meal 93.4 38.5 37.2 of inoculum at two different occasions (runs; at least 2
Rapeseed meal 92.2 39.4 27.9
Fish meal 79.3 68.2 — wk apart).
Whole cottonseed 96.2 21.4 45.0 At the end of the 48-h incubation period, tube and
1
Mixture 1471 Matmor Ltd., Ashdod, Israel. vessel contents were acidified by adding 6 M HCl to
reach a final pH of 1.3 to 1.5. After a few seconds, when
the foam subsided, pepsin powder (EC 3.4.23.1) was
added to a final concentration of 0.2% (wt/vol). The
study (Table 1). Two methods for measuring IVDMD tubes and the vessels were reincubated for an addi-
were compared: the traditional in vitro method (TT) tional 48 h. At the end of this stage, the bags were
and DAISYII system. The TT method reagents were rinsed thoroughly with tap water until the rinse was
used for both systems. All feeds were ground in a Wiley
mill (Arthur H. Thomas, Philadelphia, PA) to pass a 2-
mm pore-size screen, except whole cottonseed (linted), Table 2. Composition of diet and concentrate pellets given to cows
which was manually cracked, resulting in a large parti- and sheep.
cle size (>2 mm). % of DM1
The donor animals, two Merino rams and two Israeli
Grain
Holstein dairy cows, were fitted with a ruminal cannula Ingredient TMR mixture
(inner diameter 10 cm). The cows were in midlactation
Corn grain 21.7 17.0
and consumed a TMR formulated to meet their mainte- Barley grain 10.9 30.0
nance and production requirements (Table 2; NRC, Oat grain 10.0
1989). The TMR contained a 35:65 ratio of forage:con- Soybean meal 4.4 2.0
Rapeseed meal 4.4 5.5
centrate with 17% CP, 31% NDF, and 1.72 Mcal NEL Sunflower meal 3.2 5.0
(on a DM basis). The rams were maintained on diets Corn gluten meal 1.3 3.0
sufficient for their maintenance allowance, which con- Whole cottonseed 6.8 10.0
Wheat silage 8.0
tained a 50:50 chopped alfalfa hay and grain mixture Corn silage 19.8
(Table 2, 16% CP, 29.1% NDF, and 1.6 Mcal NEL on a Wheat bran 6.9 10.0
DM basis, mixture 1471, Matmor Ltd., Ashdod, Israel). Citrus pulp 3.6
Pea hay 3.1
The composition of the grain mixture offered to sheep Oats hay 3.1
contained ingredients similar to the TMR. The TMR NPN (mixture)2 0.5
Soybean oil 0.1 4.5
was offered once daily at 0900 h, while the rams’ meal Salt/calcium 1.6
was offered twice daily at 0900 and 1700 h. Rumen Fish meal 0.4
digesta was collected before the morning meal from the Vitamins and minerals3 0.18 3.0
reticulum near the reticuloomasal orifice by vacuum 1
TMR = Diet given to cannulated cows and grain mixture 1471,
pump. The digesta from each animal was filtered Matmore Ltd., Ashdod, Israel, given to rams.
2
through eight layers of gauze cloth, mixed on a volume Contained 80% urea and 20% ammonium sulfate.
3
basis for each species, purged with CO2, and kept in a Contained 20,000,000 IU of vitamin A/kg, 2,000,000 IU of vitamin
D/kg, 15,000 mg of vitamin E/kg, 6000 mg/kg of Mn, 6000 ppm of
prewarmed thermos until use (within approximately Zn, 2000 mg/kg of Fe, 1500 mg/kg of Cu, 120 mg/kg of I, 50 mg/kg
20 min). of Se, and 20 mg/kg of Co.

Journal of Dairy Science Vol. 83, No. 10, 2000

 IN VITRO DIGESTIBILITY METHODS 2291

clear. The tubes were centrifuged at 2500 × g for 15 Table 3. Means of in vitro DM digestibility (percentage) values using
two methods: traditional method (TT) and DaisyII 1 for different feed-
min, and the supernatant was discarded. To the pellet, stuffs.
50 ml of H2O was added and the tubes were recentri-
Main effect3
fuged to wash out the residual acid. The tubes con-
taining the pellets and the bags were dried in a forced- Feedstuff2 TT DaisyII Method
air oven at 60°C for 48 h to determine the residual DM X SD X SD P<
weights. For both methods, IVDMD was calculated as Forages
the DM which disappeared from the initial weight in- Grass hay 61.3 10.5 58.5 5.1 NS4
serted into the bag or tube. Alfalfa cubes 48.3 3.4 58.4 3.6 0.03
Alfalfa hay 58.0 5.5 53.6 4.6 NS
Clover hay 54.8 6.7 46.7 5.0 NS
Statistical Analyses Concentrates
Corn grain 92.2 14.9 85.9 11.3 NS
Results were analyzed using the general linear model Barley grain 68.1 5.9 79.1 4.1 0.02
Wheat grain 79.8 10.8 89.1 7.6 0.01
procedure of SAS (1985). The linear model used in- Sorghum grain 80.0 13.9 77.2 6.8 NS
cluded the main effects of run, inoculum source (sheep Concentrate mix5 71.6 1.5 74.4 1.3 0.05
vs. cow), the IVDMD method (TT vs. DAISYII), their Wheat bran 67.9 12.5 63.4 2.5 NS
CP supplements
interaction, and the residual error term. There were no Soybean meal 77.5 4.7 76.3 2.0 NS
interaction effects, so the model was reduced to include Sunflower meal 54.0 4.8 62.8 3.7 0.00
the main effects and the error term. Means were consid- Corn gluten feed 66.6 6.4 70.0 3.4 NS
Cottonseed meal 56.6 13.4 54.8 3.5 NS
ered significantly different at P< 0.05. Rapeseed meal 66.5 2.9 75.0 4.2 0.001
Fish meal 81.3 2.1 91.0 0.5 0.001
Whole cottonseed 36.5 5.7 38.9 3.7 NS
RESULTS
1
ANKOM Technology Corporation, Fairport, NY.
Chemical Analysis of Feeds n = 12.
2
3
Method: TT versus DaisyII. There was no significant effect of ru-
The chemical composition of the feeds tested (Table men inoculum source (sheep vs. cow) or run.
1) compared well with previous reports from our labora- 4
NS = Not significant (P < 0.05).
tory (Mabjeesh et al., 1997; Shabi et al., 1998; Arieli et 5
Mixture 1471, Matmore Ltd., Ashdod, Israel.
al., 1999) and others (NRC, 1989; Holden, 1999). The
CP content of the CP supplements ranged from 30% in
the corn gluten feed to 68% in the fish meal. The CP The IVDMD values differed between the TT and
content of the concentrate grains ranged between 9 and DAISYII methods for some feeds from each group. The
14%, the highest being wheat grain. The CP content of IVDMD of alfalfa cubes was 21% higher in DAISYII
roughages ranged from 22 to 11%, the highest being than in the TT method. The grains exhibited similarity
alfalfa hay. Whole cottonseed was not included in any between the two methods for corn, sorghum, and wheat
of the feedstuff groups because of its relative high con- bran (averaging 89, 79, and 66%, respectively). Barley
tent of CP and fiber (>18%). The NDF content of the grain, wheat grain, and the concentrate mix had higher
feeds ranged from 11 to 58%, the highest being in the IVDMD values with the DAISYII method than with
roughages, intermediate in the CP supplements, and TT. The same trend was apparent in some of the CP
lowest in the grains. The OM content ranged from 79 supplement feeds. Sunflower meal showed the highest
to 97%, the lowest being for fish meal. difference at +17% from DAISYII relative to TT, versus
+13% and +12% for rapeseed and fish meal. Whole cot-
IVDMD tonseed had the lowest IVDMD (38%) relative to all
feed categories.
The IVDMD values are presented in Table 3. The
IVDMD of roughage feeds ranged from 47 to 61%, being DISCUSSION
highest for grass hay and lowest for clover hay. The
IVDMD value for the grains ranged from 63 to 92%, The in vitro method of evaluating the digestibility of
being highest for corn. The CP supplements also exhib- ruminant feeds is used worldwide. The method is easier
ited a wide rage of IVDMD values, 55 to 91%, being than in vivo analysis and avoids the need to surgically
lowest for cottonseed meal and highest for fish meal. prepared animals in different locations in the gastroin-
The IVDMD of whole cottonseed was low relative to the testinal tract. The TT method has also been proven
other feedstuffs, averaging 38%. The IVDMD values more accurate than digestibility predictions based on
of all feedstuffs were similar for the two sources of the chemical compositions of feeds (Van Soest, 1994).
inoculum: sheep and cow (data not shown). The TT method was first introduced to predict forage

Journal of Dairy Science Vol. 83, No. 10, 2000

2292 MABJEESH ET AL.
digestibility and later modified for starchy feedstuffs.
Because of its similarity to in vivo values (Tilly and
Terry, 1963; Aufrere and Michalet-Doreau, 1988), it
is considered a reference method for the prediction of
ruminant feed digestibility. In this study, we compared
the DAISYII method to the TT method with two sources
of rumen inoculum, focusing on the reliability of the
DAISYII method for predicting digestibility of differ-
ent feedstuffs.
The source of the inoculum did not affect the IVDMD
of any of the feedstuffs used in this study. Cone et al.
(1989) found that the type of diet fed to the donor animal
affects the values of in vitro degradability. The starch
degradability from different feedstuffs was higher when
the donor cow was fed a ration containing 1:1
hay:concentrate (on a DM basis) than when the diet
was based solely on hay. However, the composition of
the concentrate mixture had only a minor effect on
degradability values (Richards et al., 1995; Stern et al.,
1997). In the current study, both sheep and cows appear
to have consumed diets that allowed sufficient growth
of similar mixed microflora in both species. The level
of feeding may also affect activity in the rumen liquor.
Cone et al. (1989) showed that the activity of rumen
microflora, measured at different times after feeding,
was higher in liquor taken from cows fed a higher level
of DM when feeds were incubated for 6 h. Since this
effect is time-dependent, it might be irrelevant to the
extent of the TT and DAISYII measurements of feed
digestibility. Similarly, others (Goldman et al., 1987)
had shown a negligible effect on the IVDMD values of
feeds, whether rumen liquor was taken from heifers,
dry cows, or lactating cows (>30 L of milk/d).
In general, the IVDMD values obtained in this study
were within the range reported by the NRC (1989) for
the TDN of these feeds. Moreover, the IVDMD values
compared well with those measured by the TT method
(Aufrere and Michalet-Doreau, 1988). However, the
barley, soybean meal, and gluten feed IVDMD values
observed in this study by the TT method were somewhat
lower than the those reported by Aufrere and Michalet-
Doreau (1988) of 83, 90, and 81%, respectively. This
discrepancy could be due to the method of commercial
feed processing and the different chemical compositions
of the feeds. The DAISYII system IVDMD values for
alfalfa hay and corn grain compared well with the re-
sults reported by Holden (1999) using the same method.
It should be noted that the IVDMD value of whole cot-
tonseed in this study was much lower (40%) than the
one reported in NRC tables (1989). This lower value
can be explained by the way cottonseed was prepared
in our study for in vitro incubation: the whole cotton-
seed was manually cracked with pliers to prevent the
extraction of oil, which could mix with the lint; if the
Journal of Dairy Science Vol. 83, No. 10, 2000
cottonseed had been ground in a Wiley mill, such mixing
would have made obtaining a homogeneous sample im-
possible.
In general, relative to TT method, the DAISYII system
gave similar IVDMD values for roughages and higher
values for concentrates and some CP supplements. The
IVDMD determined by the DAISYII method might be
affected by several factors related to the bags used, feed
characteristics, and the instrument itself. One obvious
advantage of the DAISYII instrument over the TT
method is the continuous rotation of the fermentation
vessels, which thoroughly mixes the digestion inoculum
during the incubation period and eliminates the need
for a time-consuming centrifugation step at the end of
incubation. On the other hand, the porosity of the bags
and the sample weight per bag surface area may affect
the IVDMD values. These factors have been extensively
reviewed for the in situ method (Nocek, 1998; Vanzant
et al., 1998). However, the repeatability of measure-
ments in DAISYII seems to be higher, as indicated by
overall lower SD across all feeds measured (Table 3).
The pore size of the bags (50 ± 15 µm) was within the
range summarized by Vanzant et al. (1998) for many
studies reported in the literature. The ratio of sample
size to bag surface area was 8.8 mg/cm2, similar to that
suggested (10 mg/cm2; Vanzant et al., 1998) to increase
the accuracy of degradability predictions relative to in
vivo ruminal disappearance. However, in this study
IVDMD values measured by the DAISYII method were
consistently higher than those obtained with TT. This
could be related to the physical properties of the feeds
used in this study. Higher IVDMD values were ob-
served in the concentrate and CP supplement groups.
These observations may be explained partially by the
fact that grains become floury when ground and by the
protein solubility versus degradability in CP supple-
ments. Sample processing, particularly grind size, in-
teracts with pore size of the bag and affects the extent
of feed disappearance. Most of the differences in re-
sponse to grind size are explained by differences in
particle size and distribution (Michalet-Doreau and
Cerneau, 1991; Michalet-Doreau and Ould-Bah, 1992;
Norzière, 1996). Moreover, Malestein et al. (1982) sug-
gested that simultaneous incubation of different
starchy feedstuffs might enhance the degradation of
separate feedstuffs. This is unlikely to be the case in the
current experiment. However, in the DAISYII system,
incubation of different feeds in the same vessel has
been shown not to affect the IVDMD values for the
different feeds (Holden, 1999).
The effects of size distribution of feed particles may
not be the sole reason for the higher IVDMD values
with the DAISYII system. It is worth noting that these
effects are more critical in the in situ method because
 IN VITRO DIGESTIBILITY METHODS
the disappearance of nutrients in a particular feed is
measured on a time function, which in turn affects de-
gradability rate. However, the time criteria for both TT
and DAISYII are irrelevant because the 48-h incubation
time for the two stages is considered to be sufficient
for complete digestion of feed matter (Tilly and Terry,
1963). Hence, the higher values observed in the DAISYII
method might be related to the nonrecovered material
that escaped from the bag and was not digested by the
two stages of the method. This phenomenon was very
well demonstrated by Dewhurst et al. (1995), who com-
pared the extent and rate of OM digestibility measured
in situ with those determined in vitro. They observed
that feeds like soybean hulls and corn germ have higher
OM digestibility in situ than in vitro because of the
passage of small particles of unfermentable material
through the bag pores in the former method. For exam-
ple, the fish meal in this study contains ∼20% ash and
other components (e.g., bones and shells), part of which
might not be water-soluble but might escape from the
bags during incubation. Furthermore, the method of
meal processing may also create insoluble compounds
(e.g., Maillard reactions) whose particle sizes may be
suitable for escape from the bags during incubation but
which are not available to the microbial enzymes or
pepsin hydrolysis. This might also be the case with the
alfalfa cubes, which had a lower IVDMD value in the
TT than alfalfa hay, while in the DAISYII method the
IVDMD was 21% higher than the TT values for alfalfa
cubes only. During the preparation of alfalfa cubes,
freshly cut plants are heated to dryness, which appears
to produce indigestible material. This is supported by
the higher ADIN content measured in the alfalfa cubes
versus the alfalfa hay (2.24 and 1.60 mg N/g of DM,
respectively). This difference between the two methods
was not observed in this study (1999) for any of the
feedstuffs used. This could be partially related to the
large apparent variation between and within the
means, especially for the grains group.
When IVDMD obtained from TT was plotted against
the DAISYII data, the following linear equation was ob-
tained:
IVDMDTT = 0.84(±0.10)IVDMDDAISYII + 8.88
(±7.24), P < 0.00001, n = 17, SD = 6.24, R2 = 0.81.
This SD of the regression and SE of the intercept and
slope point to a large scatter around the regression line.
However, when the data from the feeds that had higher
values in the DAISYII method were excluded from the
regression analysis, the R2 improved to 0.95 and the
following regression was calculated:
2293
IVDMDTT = 1.02(±0.08)IVDMDDAISYII + 1.11
(±5.43), P < 0.00001, n = 10, SD = 3.76.
This regression shows that the DAISYII method has
good potential to predict digestibility of certain feed-
stuffs. Because the difference between the methods is
mainly related to the particle size of the indigestible
material in the feeds, we suggest that these groups of
feed be ground to a larger particle size, which may
prevent the escape of indigestible particles from these
components from the bags.
CONCLUSION
The DAISYII system is an easier, less time-consuming
method of measuring IVDMD of ruminant feed than
the method of TT. However, its use in the current proce-
dure is limited to feeds that do not contain a high level
of small indigestible derivatives that might escape from
the bags during the incubation. Rumen liquor taken
from sheep maintained on a diet with a 1:1 forage:con-
centrate ratio was as efficient for IVDMD measure-
ments as inoculum taken from the rumen of dairy cows.
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