Livestock Production Science 97 (2005) 219 – 229
www.elsevier.com/locate/livprodsci
Physico-chemical characteristics and degradation rate of soluble
protein obtained from the washout fraction of feeds
M. Gierus*, L. de Jonge, G.A.L. Meijer
Nutrition and Food Division, Animal Sciences Group, Wageningen University and Research Center,
P.O. Box 65, NL 8200 AB Lelystad, The Netherlands
Received 16 November 2004; received in revised form 14 April 2005; accepted 2 May 2005
Abstract
The (water)-soluble fraction of feeds is often assumed to be completely and immediately degraded in the rumen. The
objective of this study was to separate the washout fraction (fraction A) obtained usually by difference after submitting the
nylon bags to the machine-washing program, to investigate the nature and the degradation of the soluble crude protein in the
washout fraction. The washout fraction obtained in vitro (filtrate) of 10 feeds was collected in water using nylon mesh as a filter.
The feeds used in the study were: two grass silages, soybean meal, three corn gluten feeds, lupine meal, rapeseed meal, wet
brewers grain silage and corn gluten feed silage. Average N losses during filtration and from machine-washed nylon bags
ranged from 15% (rape seed meal) to 74% (grass silage) and were not different between procedures. N recovered in the filtrate
ranged from 12% (soybean meal) to 60% (corn gluten feed silage) of sample N. The three fractions obtained from the filtrate
were: soluble protein (TP), non-protein N (NPN) and fine particles (NS). The NS fraction was obtained after centrifugation of
the filtrate and comprised 0% to 87% of N in the filtrate. Soluble protein (TP) in the supernatant was obtained after precipitation
with trichloroacetic acid and N in the remaining supernatant was defined as non-protein N (NPN). Significant amounts of TP
were found in soybean meal (58%), lupine meal (30%) and rapeseed meal (27%) as percent of total N in the filtrate. NPN
ranged from 13% to 100% of N in the filtrate. The in vitro incubation of the protein N (NS + TP) showed that all fractions were
not completely degraded, suggesting a potential participation as escape protein. Fine particles in the filtrate have similar
degradation rates as the residue left in the filter. It is concluded that the washout fraction consisted of different crude protein
fractions that were not always completely and immediately degraded in the rumen.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Nylon bag; Washout fraction; Trichloroacetic acid; Soluble protein degradation
1. Introduction
* Corresponding author. Present address: Grass and Forage Science/Organic Agriculture, Institute of Crop Science and Plant
Breeding, Christian-Albrechts-University of Kiel, 24098 Kiel, Germany. Tel.: +49 431 880 1662; fax: +49 431 880 4568.
E-mail address: [email protected] (M. Gierus).
0301-6226/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.livprodsci.2005.05.002
In dairy cows, alpha-amino acids available for
absorption in the small intestine originate largely
from microbial protein and ruminally undegraded
220
M. Gierus et al. / Livestock Production Science 97 (2005) 219–229
feed protein (UDP). The estimation of protein degradability and protein escape are necessary for an adequate diet formulation. The degradability of dietary
proteins in the rumen as well as the estimation of
escape protein is often assessed using the nylon bag
technique. This technique plays a central role in several protein evaluation systems for dairy cows. In the
nylon bag technique, the fraction A (Mehrez and
Ørskov, 1977), which may contain soluble protein
and protein in fine particles (e.g., b40 Am), leaves
the bags. In most protein evaluation systems, this
fraction A is assumed to be immediately and completely degraded in the rumen (AFRC, 1993; Tamminga et al., 1994; Madsen et al., 1995). This implies
that only insoluble protein can leave the rumen. However, this assumption may be incorrect. Although the
estimation of undegraded protein in the Dutch DVE/
OEB and French PDI system use the factor 1.11 to
correct the amount of undegraded feed protein, the
results of Van Vuuren et al. (1998, 2000) showed that
the estimation of undegradable protein fraction in
some feeds, such as wet brewers grains could be
higher than assumed by the system. This could be
explained by the fact that the washout fraction (fraction A) is not, in most cases, immediately and completely degraded in the rumen (Messman et al., 1994;
Gierus et al., 2001). The washout fraction can be
rather substantial for some feeds. For example, Madsen et al. (1995) reported a washing loss of N from
bags ranging from 33.3% to 47.3% of the total N
content for different concentrate feeds. Based on
such results, Weisbjerg et al. (1990) suggested a correction for fine particle losses from the nylon bag,
assuming the particle loss is degraded in a way similar
to that of the remaining feed in the bag. However,
besides fine particles, the amount of N washed out
from the nylon bag may contain soluble protein,
which may be resistant to rumen degradation and
therefore, contribute to the escape protein. Thus, the
rumen escape protein fraction estimated by nylon bag
technique is underestimated.
Because the in situ technique does not account
differently for the N compounds in the fraction A,
nor can it be used to estimate the degradation rate of
fraction A, the present study had two objectives: (1) to
characterize and quantify the different N compounds
in the washout fraction of 10 feeds and (2) to examine
to what extent the fine non-soluble particle (NS) and
trichloroacetic acid-precipitable proteins (TP) in the
washout fraction were degraded using an in vitro
method (Broderick, 1987). Consequently, the extent
to what these fractions contribute to the feed protein
escaping ruminal degradation was estimated.
2. Materials and methods
2.1. Sample description and preparation
Ten feeds of different origin and characteristics
were utilized. Six concentrate feeds, two wet by-products and two samples of grass silage were used:
soybean meal (SBM), lupine meal (LPM), rapeseed
meal (RSM), three corn gluten feeds (CGF-1, CGF-2
and CGF-3), silage of wet brewers grain (WBGS) and
silage of corn gluten feed (CGFS). The WBGS
contained 5% of beet pulp (DM basis). Grass silages
(GS-1 and GS-2) were taken from the sample collection of the Nutrition and Food Division in Lelystad
(The Netherlands). They differed in DM content, as a
result of different wilting times (Table 1). Both silages
originated from a plot fertilized with 300 kg N/ha/year
at the regional bCranendonckQ experimental farm at
Maarheeze (The Netherlands) and were harvested on
June 17th, 1997 (second cut). The first silage was
grass wilted to 25% DM; the second silage on grass
wilted to 45% DM. The CP levels in these silages
were high (Table 1), but not unusual for this level of N
Table 1
Chemical analyses and feed evaluation of the 10 feedsa
Feeds
DM,
%
CP,
g/kg
DM
Fat,
g/kg
DM
Ash,
g/kg
DM
Starch,
g/kg
DM
DOM,
% of
OM
Grass silage 1
Grass silage 2
Soybean meal
Corn gluten feed 1
Corn gluten feed 2
Corn gluten feed 3
Lupine meal
Rape seed meal
Wet brewers grain
silage
Corn gluten feed
silage
25.1
52.9
87.8
89.3
90.1
93.5
91.7
89.6
28.2
295
288
544
227
209
199
322
344
221
NA
NA
21
37
21
53
58
32
100
113
108
72
58
58
135
27
72
52
NA
NA
ND
173
149
124
ND
ND
NA
80.3
79.5
90.9
86.5
86.5
82.5
89.1
75.9
59.0
45.3
161
20
43
316
88.9
a
DM = dry matter, CP= crude protein, DOM = digestible organic
matter, NA= not analyzed, ND = not detected.
M. Gierus et al. / Livestock Production Science 97 (2005) 219–229
fertilization and harvesting date (Hindle et al., 2000).
The grass had been chopped into 3 cm pieces for the
present analysis. Because CGF-1 and CGF-2 were
delivered in the form of pellets, they were milled
through a 3 mm screen. Other samples were not
treated prior to the study. All dry feeds were stored
in a refrigerator at 4 8C and silages were stored at
12 8C.
2.2. Machine washing of nylon bags
As reference for the filtration procedure, the size of
the washout fraction was determined using a conventional machine washing (AEG Turnamat 2800) of
nylon bags (Nybolt 30/40) according to Hindle
(1994) in four replicates of all 10 feed samples. The
amount of water used was about 40 l and the centrifugation step was excluded. Each washing run took
about 45–50 min. The loss of DM and total N was
calculated from DM and the N content (Kjeldahl-N)
of the original feed samples and the residues after
washing and drying at 70 8C for at least 12 h. Total
N loss during washing was assumed to be the difference in total N content of the feed sample before
washing and the total N content of the residue after
washing.
2.3. Filtration procedure
2.3.1. Development of the filtration procedure
A set of filtration trials was conducted. Initially, the
recovery of the DM lost in the filtrate in the smallest
possible volume of water was determined. Sample
size and amount of rinsing water were varied, and
loss of DM due to filtration was estimated as the
difference between DM weight of the sample and
DM weight of the residue after filtration (Table 2).
In the first trial, lowering the amount of rinsing water
to 45 ml decreased the DM loss significantly. In the
second trial, no change in DM loss in filtration was
observed when the sample size varied within the range
of 1.2 g to 3.6 g of DM. The DM loss was, however,
significantly lower when using 5.4 g DM of sample.
2.3.2. The filtration procedure as used in the study
For the concentrate ingredients, 3–5 g of fresh
material was taken as sample size. For WBGS,
CGFS and GS-2, 4 g of fresh material were taken.
221
Table 2
The loss of DM (%) in soybean meal (SBM) as a result of filtration
using different sample sizes and amounts of rinsing water
Feed
Sample
size, g DM
Rinsing
water, ml
Loss of
DM, %
Trial 1: effect of amount of rinsing water
SBM (n = 4)
2.7
75
SBM (n = 4)
2.7
45
31.4a
27.5b
Trial 2: effect of sample size
SBM (n = 4)
1.2
SBM (n = 4)
2.7
SBM (n = 4)
3.6
SBM (n = 4)
5.4
31.7a
31.4a
31.2a
29.9b
75
75
75
75
a,b
Different superscripts within the same trial indicate significant
differences ( P b 0.05).
Because of its low DM content, the sample size for
GS-1 was increased to 5 g. Samples of each feed were
weighed into 250 ml beakers and soaked in 25 ml of
distilled water during constant shaking (Gerhardt
Schüttelmaschine RO20, Bonn, Germany) at 160
rpm/min for 1 h. Samples of GS-1 were, because of
their bulky nature, soaked in 35 ml of water. After
soaking, the samples were poured into the funnels
containing a suspended filter. This filter was a piece
of nylon mesh (41 Am pore size, Nybolt 30/40) that
was used for the in situ trials. The filtrate was collected in a 100 ml centrifugation tube. The beakers
were washed to get the remaining feed from the
beakers into the funnels and the filter with residues
placed into the oven (Hereaeus Electronics, type T50)
for drying at 70 8C for determination of DM losses
and N-content (Kjeldahl-N) of the residue.
Each filtrate was further divided into three different
fractions. The fractions considered were the fine nonsoluble particles (NS), soluble non-protein nitrogen
(NPN) and trichloroacetic acid-precipitable protein
(TP), as described in the following.
2.4. Nitrogen fractions in the filtrate
2.4.1. Fine non-soluble particles (NS fraction)
Firstly, each filtrate was centrifuged (BHG Hermple, Z 424) at 1500g for 20 min and the supernatant
was removed to a 100 ml centrifugation tube. The
pellet (pellet 1) after this centrifugation was assumed
to contain non-soluble particles and dried at 70 8C for
the determination of DM weight and N content.
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M. Gierus et al. / Livestock Production Science 97 (2005) 219–229
2.4.2. Trichloroacetic acid-precipitable proteins (TP
fraction)
Secondly, an amount of a solution of 1000 g/l trichloroacetic acid (TCA) was added to the supernatant
obtained as described above to achieve a final concentration of 100 g/l TCA in solution. The concentration of
10% TCA was used to precipitate all soluble protein in
the supernatant (Greenberg and Shipe, 1979; Marais
and Evenwell, 1982), which was confirmed in a preliminary pilot experiment on quadruplicates of filtrates
from SBM and CGF-2. The contents of the tubes were
well mixed and the tubes were left for 2 h at 4 8C to
allow complete precipitation of protein. These tubes
were then centrifuged at 1500g for 20 min. The pellet
(pellet 2), assumed to contain all water-soluble true
protein, was dried and analyzed for DM and N content.
ly and kept at 39 8C. All manipulations were performed under continuous flushing with oxygen-free
carbon dioxide.
2.4.3. Non-protein nitrogen (NPN fraction)
The supernatant after precipitation was assumed
to contain all soluble non-protein N and is defined as
non-TCA-precipitable N. This supernatant was
brought to a constant volume of 100 ml with distilled water. From this volume, an aliquot of 10 ml
was taken for N determination. The DM content of
the supernatant (DMSUP) was calculated by difference as follows: DMSUP = DMS DMRES DMNS DMTP, where DMS, DMRES, DMNS and DMTP are
the DM content in the sample, in the residue remaining after filtration, in the NS fraction and TP fraction, respectively. The pellets and the supernatants
were stored at 4 8C prior to analysis.
2.5.3. Sample preparation
Feeds used in this experiment were 5 out of 10
used before: LPM, CGF-2, CGFS, SBM and WBGS.
The feeds were filtered according to the procedure
described previously in order to obtain enough N in
each fraction both for N determination and for the in
vitro study. Four fractions of each feed were obtained
for the study, which correspond to the fractions as
obtained by filtration: (a) total filtrate (FIL), representing NPN, TP and NS, i.e., the washout fraction; (b)
supernatant (SUP), representing the TP and NPN in
the washout fraction; (c) pellet (PEL), representing the
NS in the washout fraction; and (d) residue (RES),
which remains after filtration, representing the potentially degradable fraction of feeds, including the indigestible fraction C from the in situ method. Fractions
with less than 5% of the sample N content in anyone
of the fractions were not considered for further analysis, as it was the case with the PEL fraction of SBM.
After performing the filtration procedure, samples of
each fraction were freeze-dried and an aliquot removed for N content determination. Samples of the
RES were milled to pass through a 1 mm mesh. Other
fractions were used without milling.
2.5. Protein degradation in vitro
An inhibitor in vitro system, based on Broderick
(1987), was used with few modifications as described
below.
2.5.1. Preparation of rumen fluid
Rumen fluid was obtained 1 h after the morning
feeding from two non-lactating cows. The cows received a standard diet of 9 kg hay fed twice daily
(2 4.5 kg) and 1 kg concentrate fed once a day.
Rumen fluid from both cows was combined and
strained through nylon filter cloth of 40 Am. Equal
amounts of McDougall’s buffer and the strained
rumen fluid were poured into a pre-heated fermentor
to obtain the inoculum, which was stirred continuous-
2.5.2. Pre-fermentation
The pre-fermentation steps were performed within 3
h and consisted of the addition of a carbohydrate mixture to decrease the ammonium concentration in the
inoculum and to increase microbial activity. The carbohydrate mixture was based on maltose, starch, xylose
and pectin, and 7.0, 3.5, 3.5 and 3.5 g/l of inoculum was
added, respectively. Pectin was dissolved in 75 ml of
warm McDougall’s buffer overnight. The inoculum
was sampled at the start and every hour thereafter to
monitor the pH. If necessary, a 3 N NaOH solution was
used to adjust pH to 6.4 in the inoculum.
2.5.4. Incubation
Samples of each fraction were weighed into 250 ml
flasks in order to ensure a sample N content of 0.20
mg/ml. Before starting the incubation, samples of the
fractions were soaked in 30 ml McDougall’s buffer at
39 8C for 1 h. Twenty minutes before the incubation
M. Gierus et al. / Livestock Production Science 97 (2005) 219–229
started, inhibitors were added to the inoculum (0.2 g
hydrazine sulfate, 0.5 g chloramphenicol and 310
Al mercaptoethanol per liter of inoculum) as described
by Broderick (1987). The incubation was started by
adding 120 ml of inoculum into the pre-heated incubation flasks. The flasks were flushed with oxygenfree carbon dioxide and sealed with Bunsen valves.
Each flask represented a whole incubation set, with
sampling times at 0, 180 and 240 min. Three flasks
were used per feed fraction. Casein and bovine serum
albumin were used as controls in each run. Flasks were
placed into a water bath and shaken continuously at
150 rpm. At each sampling time, 10.0 ml of the
inoculum was collected and transferred to centrifuge
tubes, previously placed in ice and containing 2 ml of
65% (w/v) of trichloroacetic acid solution. Tubes were
kept at least 15 min on ice before centrifugation at
3000g for 20 min at 4 8C. Nitrogen was determined
in the supernatant and assumed to be NPN.
2.5.5. Calculations
The amount of TP incubated was determined from
the amount of non-TCA-precipitable N at zero incubation time by difference. For each sampling time, undegraded N content was calculated as a proportion of the
total amount of incubated sample N after subtracting
the N content of blank flasks at each incubation time.
Degradation rate (k d4) was computed as:
h1 ¼ ðlnB4 lnB0 Þ=4 h
where B 0 and B 4 are the amount of TP after 0 and 4
h incubation time.
Estimated protein N escape was computed as:
g kg1 total N ¼ B0 kp = kp þ kd
223
samples were analyzed for non-TCA-precipitable N
as performed by Neutze et al. (1993). Degraded N
corresponded to the appearance of TCA-soluble N
(NPN) in the supernatant after incubation, corrected
for the TCA-soluble N content in the inoculum
(blank tubes).
Feeds were sampled and analyzed for DM, CP
(Kjeldahl-N 6.25), crude fat, ash and starch, as
described by Steg et al. (1990). Organic matter digestibility (DOM) was established in vitro (Tilley and
Terry, 1963). The results of these chemical analyses
are in Table 1.
2.7. Statistical analysis
Student’s t-tests were used to compare the effect of
filtration and machine washing on DM and N content
in residues. Fractions obtained by the separation procedure for each feed were submitted to analysis of
variance and means separated by Tukey’s test at
P b 0.05. Due to the presence of zero values, data
was transformed as ( y + 0.5)0.5 for statistical analysis
(Steel and Torrie, 1980). For the degradation study,
fractions within feeds were submitted to analysis of
variance and means separated by Tukey’s test at
P b 0.05.
3. Results
3.1. Filtration procedure
where k p is the ruminal passage rate and was estimated assuming a passage rate of the solid phase
ranging from 2% to 8% h 1 and a passage rate of
the liquid phase of 12% h 1. Undegraded fractions in
RES were not corrected for the undegradable fraction
C as well as fractions were not corrected for ammonia N contamination.
3.1.1. Nitrogen losses
The percentage of N remaining in the residue after
machine washing ranged from 25.8% to 84.4% for
GS-1 and SBM, respectively (Table 3). A similar
range (24.2% to 89.1%) for the same feeds was
obtained using the filtration procedure. The differences in %N in residues between the two methods
were in the range of 5.5% (WBGS) to 10.6% units
(CGFS) for all feed, and were significant for WBGS,
CGFS and CGF-2. The difference between methods in
N loss was the highest for CGFS ( P b 0.001).
2.6. Chemical analysis
3.2. Nitrogen in different fractions after filtration
The total N content of feeds and fractions were
determined by the Kjeldahl method (ISO, 1991). The
The amount of N recovered in the different fractions of the filtration procedure is presented in Table
224
M. Gierus et al. / Livestock Production Science 97 (2005) 219–229
Table 3
Kjeldahl-N in residue, as % of N in sample, after machine washing
and filtrationa
Grass silage 1
Grass silage 2
Soybean meal
Corn gluten feed 1
Corn gluten feed 2
Corn gluten feed 3
Lupine meal
Rape seed meal
Wet brewers grain
silage
Corn gluten feed
silage
%N in residue
Contrasts
Machine Filtrationb (F)
washing Series Series
(M), % 1, %
2, %
F1–F2
SED
M–F
SED
25.8
48.0
84.4
52.9
37.2
43.2
61.5
82.1
58.6
27.8
50.2
81.3
54.4
39.8
44.6
56.0
83.5
63.5
24.2
48.2
85.4
53.5
39.5
44.3
61.1
89.1
70.1
0.4***
1.3
0.4***
1.2
0.2
1.3
2.2
1.6*
1.3**
1.1
1.0
1.1
0.8
0.2***
0.9
2.0
1.9
2.0**
48.3
33.9
31.0
4.2
2.9***
a
All values are averages of four replicates.
Filtration 1: day 1 and 2: day 2.
* P b 0.05.
** P b 0.01.
*** P b 0.001.
b
4. The recovery of N from the sample in the different
fractions ranged from 80% for GS-1 to 103% for
LPM. The amount of N in the washout fraction (filtrate) of feeds ranged from 9.0% (RSM) to 60.5%
(CGFS). The washout fraction formed a considerable
part of the feeds total N content for all of the feeds
except for SBM, RSM and WBGS. However, the
latter feeds are rich in N; thus, the absolute amount
of N in the washout fraction is not negligible.
For GS-1, GS-2, CGF-1, CGF-2, CGF-3 and
CGFS, the washout fraction consisted mainly of
NPN, ranging from 80% (CGFS) to 100% (GS-1) of
the filtrate. Significant amounts of TP were only
present in LPM (12.4%), SBM (7.0%) and RSM
(2.4%), as percentage of N in the sample. WBGS
and LPM were distinguished by having substantial
amounts of NS in the washout fraction, i.e., 22.4%
and 21.8%, respectively (Table 4).
3.3. Degradation of crude protein in the different
fractions
The amount of potentially degradable N incubated,
i.e., TCA-precipitable N (TP) varied from 9.0 mg/g
DM in the PEL fraction of CGFS to 118 mg/g DM in
the PEL of LPM (Table 5). Depending on the feed, the
fraction SUP has the lowest content of TP among
fractions, contributing negatively to the accuracy of
calculation of protein degradation.
After the 4 h incubation period, considerable degradation was observed in all fractions. However, undegraded N was present after 4 h in all feeds analyzed,
allowing to estimate the escape protein for each fraction. At k p of 4% h 1, escape protein estimates varied
from 3.31% to 42.54% in FIL fraction, from 1.19% to
14.11% in SUP fraction, from 19.99% to 60.43% in
PEL fraction and from 18.51% to 74.87% in RES
fraction. In WBGS, only small amounts were degraded after 4 h incubation period, with high proportions
of escape protein, especially RES fraction. Although
results for all feeds analyzed are presented for the
escape protein of RES at 12% h 1, the largest proportion are expected to have a passage rate compatible
with the solid phase.
RES and PEL showed similar degradation rate for
the feeds analyzed. The degradation rate after 3 h incubation time showed less variation than the degradation computed after 4 h incubation time. Nevertheless,
Table 4
Nitrogen in residue after filtration, and in the non-soluble fraction
(NS), the TCA-precipitable fraction (TP) and the non-protein nitrogen fraction (NPN) as percentage of nitrogen in the sample
Grass silage 1
Grass silage 2
Soybean meal
Corn gluten feed 1
Corn gluten feed 2
Corn gluten feed 3
Lupine meal
Rape seed meal
Wet brewers grain
silage
Corn gluten feed
silage
S.E.
Residue
(%)
Filtrate
NS*
(%)
TP*
(%)
NPN
(%)
24.3h
48.1de
85.5a
53.7d
39.4f
44.4ef
61.1c
89.2a
69.9b
0g
0.9f
1.9e
4.6cd
5.7c
3.4d
21.8a
1.1ef
22.4a
0d
0d
7.0b
0.3d
0.7d
0d
12.4a
2.4c
0d
56.1a
41.3d
3.1f
39.0d
51.0b
47.1c
7.6e
5.5ef
3.5f
80.5d
90.3c
97.1abc
96.4abc
96.6abc
93.9bc
102.9a
98.3ab
96.0abc
30.5g
12.0b
0d
48.5bc
91.0bc
2.8
1.1
0.6
2.1
1.2
Recovery
(%)
Means within a column without a common superscript differ
( P b 0.05).
* Due to the presence of zero values, data was transformed as
( y + 0.5)0.5 for statistical analysis (Steel and Torrie, 1980).
M. Gierus et al. / Livestock Production Science 97 (2005) 219–229
Table 5
Protein degradability and percent escape protein of feed samples
and fractions
Feed N content TP
Deg.
Escape, %
in fraction,
rate, h 1
mg/g DM
B 0, % B 4
0.02* 0.04
0.08
0.12
Lupine meal
FIL 61.9
SUP 45.7
PEL 118.0
RES 46.6
S.E.
80.5b
61.4c
94.4a
92.6a
1.6
0.210a
0.333a
0.149a
0.160a
0.07
7.01c
3.48d
11.18a
10.29b
0.10
12.89c
6.60d
19.99a
18.51b
0.21
22.22c
11.91d
33.00a
30.86b
0.36
29.29c
16.29d
42.13a
39.68b
0.49
Soybean meal
FIL 25.3
SUP 23.9
PEL ND
RES 103.0
S.E.
66.0b
61.0c
ND
99.8a
0.42
0.184a
0.133ab
ND
0.056b
0.02
6.48c
7.97b
ND
26.40a
0.07
11.80c
14.11b
ND
41.78a
0.01
20.02c
21.91b
ND
58.93a
0.02
26.07c
28.94b
ND
68.28a
0.20
0.114a
0.551a
0.054a
0.047a
0.17
2.54c
0.47d
23.90b
26.87a
0.19
4.42c
1.19d
37.62b
41.38a
0.31
7.02c
2.18d
52.75b
56.69a
0.46
8.73c
3.02d
60.91b
64.67a
0.55
Wet brewers grain silage
FIL 40.6
63.0b
SUP 26.2
26.8c
PEL 81.6
89.8a
RES 31.1
89.2a
S.E.
3.64
0.019b
0.173a
0.019b
0.008b
0.02
32.12c
2.79d
45.54b
64.50a
1.80
42.54c
5.05d
60.43b
74.87a
2.38
50.78c
8.50d
72.24b
81.41a
2.84
54.29c
11.00d
77.28b
83.85a
3.05
Corn
FIL
SUP
PEL
RES
S.E.
0.138a
0.385a
0.068a
0.044a
0.11
1.87c
0.63c
17.61b
28.59a
0.34
3.31c
1.19c
28.70b
43.60a
0.53
5.41c
2.18c
41.88b
59.11a
0.75
6.86c
3.01c
49.45b
67.07a
0.87
Corn
FIL
SUP
PEL
RES
S.E.
gluten feed silage
44.1
17.0b
60.5
13.5b
9.0
88.2a
14.3
90.0a
1.25
gluten feed 2
51.0
14.8c
53.9
12.7c
28.1
77.4b
20.6
91.8a
1.29
B 4: degradation rate calculated after 4 h incubation time.
ND = not determined.
FIL= filtrate fraction, corresponding to the whole fraction A as
measured in the in situ method.
SUP= supernatant, corresponding to the true protein fraction, i.e.,
TCA-precipitable fraction in the fraction A.
PEL= pellet, corresponding to the insoluble small particles in the
fraction A.
RES = residue, corresponding to the fraction B (and C) as assumed
in the in situ method.
Mean values within columns and within each feed without a common superscript differ ( P b 0.05).
* Fractional passage rate (h 1 ).
225
similar protein nature seems to be present for degradation in the rumen for RES and PEL fractions. The
SUP fraction was the fastest degraded in almost all
feeds analyzed and higher degradation rate was observed in SUP than in PEL. The FIL fraction, containing both NS and TP fraction (FIL= SUP + PEL), was
degraded depending on the highest amount of degradable CP present in SUP or PEL.
4. Discussion
The fraction A in the in situ method contains fine
particles, soluble proteins and NPN, with variable
amounts among feeds. Corrections for the loss of
fine particles are proposed (Hvelplund and Weisbjerg,
2000) for the calculation of effective degradability,
assuming a degradation rate (c) similar as to that of
the fraction B.
For all feeds, except for SBM and RSM, N in the
washout fraction was more than 25% of sample N
(Table 4). De Boever et al. (1997) measured the
washout fraction as the difference between N content
in the sample and in the residue after machine washing. They observed a high %N lost in the washout
fraction of silages of different qualities, ranging from
52.7% to 66.4%. For soybean meal, %N in the washout fraction was lower, 11.4% to 11.7%, and consistent within qualities. These results are in line with our
observations (Table 3). For ensiled wet brewers grain,
De Boever et al. (1997) found a %N in the washout
fraction of 39.6%, which was high compared to our
result (25.9%, Table 4). The N disappearance due to
machine washing of corn gluten feed, soybean meal
and grass silage observed by Van Straalen (1995) was
48%, 11% and 54% of sample N, respectively. These
results are similar to ours for CGF-3, SBM and GS-1
obtained by filtration, being 50.5%, 12.0% and 56.1%
of sample N, respectively.
The washout fraction of GS-1 was higher than that
of GS-2. A higher NPN fraction in proportion to feed
N in GS-1 accounted for the difference. The other
difference between the two grass silages was the DM
content. Wilting before ensiling is an efficient way of
reducing proteolysis in the silo. According to Hristov
and Sandev (1997), the contents of NPN were lower
in grass silage when wilted to 475 g DM/kg before
ensiling compared to when ensiled as fresh cut (250 g
226
M. Gierus et al. / Livestock Production Science 97 (2005) 219–229
DM/kg). An extended proteolysis in the silo after
ensiling might be the reason for the difference between GS-1 and GS-2, as observed also by Messman
et al. (1994).
The recovery of N after filtration was high (N 95%)
for all concentrates, but lower in the grass silages and
CGFS (80.5% to 91.0%). One reason for this could be
a loss of highly volatile NPN such as ammonia during
the filtration procedure. Another reason could be the
retention of fine particles in the filter pores.
Only LPM, SBM and RSM had a substantial
amount of TP in the washout fraction. The absolute
amounts of TP in these fractions represent up to 40, 38
and 8 g CP/kg DM in sample, respectively. In the
subsequent degradation study, TP in SUP was considered the potentially degradable protein N. TCA is
assumed to precipitate peptides with a chain length
N10 amino acids (Van Soest, 1994). Considering 10
amino acids as the TCA-cutoff, the use of TP as a
criterion for what remains undegraded might be incorrect if peptides with less than 10 amino acids are
prone to escape further degradation to ammonia in the
rumen.
Fine particles were found especially in WBGS and
LPM. The fine particles fraction of grass silage of
ryegrass from Hvelplund and Weisbjerg (2000)
accounted for 19.8% of N in sample.
The nylon bag method is widely accepted as a
method to estimate the degradability of feeds as they
occur in the rumen. However, this method does not
account for the rapid degradable fraction of feeds, i.e.,
the fraction A. Separation using buffer, saline or
detergent solutions differs in the estimation of soluble
proteins, since solubility is dependent on inherent
physical and chemical characteristics of each protein.
Solubility in water is a feature of albumins, which are
present mostly in legume seeds, leaf proteins and
dicotyledonous plants, and less in grasses (Van
Soest, 1994), which may explain the detection of
significant amounts of TCA-precipitable proteins in
SBM, LPM and RSM.
In the present work, casein and bovine serum
albumin were used in each incubation set to monitor
the degradation of feed fractions. The degradation rate
(k d) after 4 h incubation time achieved 41.5 F 0.06%
h 1 for casein and 6.4 F 0.01% h 1 for bovine serum
albumin. The estimated escapes using a passage rate
of 6% h 1 were 9.8 F 2.0% for casein and
47.6 F 5.1% for albumin. Peltekova and Broderick
(1996) found a degradation rate for casein of 0.486
h 1 after 6 h incubation time and recent work of
Broderick et al. (2004) reported a degradation rate
of casein up to 0.318 h 1 after adding different concentration of inhibitors. For serum albumin, the degradation rate is lower, due to the disulfide bridges
encountered in this protein source. Broderick (1987)
found a degradation rate of 0.065 to 0.071 h 1 after
adding different amounts of maltose in the incubation
medium. The degradation rates for casein and serum
albumin in our study are consistent with the results
found in literature.
The results presented for RES fraction show, however, a lower degradation rate in comparison to in situ
results (Broderick, 1987; England et al., 1997; Reynal
and Broderick, 2003). Values in literature report degradation rates for SBM ranging from 8% to 17% h 1.
In the present work, the degradation rate of RES in
SBM was about 5.6% h 1. Recent work of Broderick
et al. (2004) with the inhibitor in vitro method showed
a range of degradation rates varying from 0.01 to 0.12
h 1 with different concentrations of inhibitors in the
medium for solvent SBM. However, the variation
observed by these authors may be related to an insufficient inhibitor concentration to prevent microbial
uptake of the degraded N.
Based on in vivo measurements, Van Vuuren et al.
(1998) suggested that the degradation of the washout
fraction of brewers grain was incomplete. The washout fraction of WBGS consisted mainly of fine particles, which are insoluble in water. Nitrogen insoluble
in water is often concealed in fibrous structures and
thereby protected from degradation (Lindberg, 1981).
Therefore, it is reasonable to believe the NS fraction is
not degraded faster than the potentially degradable
part of the feed. For the feeds in the present study,
PEL and RES fractions did not differ in the degradation rate, either measured after 3 h or 4 h incubation
time. Because degradation also is dependent on rumen
passage rate (effective protein degradation; Ørskov
and McDonald, 1979), the PEL fraction might be
responsible for the incomplete degradation of the
washout fraction as suggested by Van Vuuren et al.
(1998, 2000). In line with this observation, De Boever
et al. (1997) measured the washout fraction of brewers
grain (by machine washing) to contain 39.6% of total
feed N, which is in accordance with our observation
M. Gierus et al. / Livestock Production Science 97 (2005) 219–229
(Table 3). A considerable part of the washout fraction
comprised non-soluble particles (washout fraction–N
soluble in borate-phosphate buffer). It was concluded
that, because of this NS fraction, protein degradation
of brewers grain measured in situ was seriously overestimated. Our results are thus in line with observations of others and suggest that the NS proteins in the
washout fraction may be the major determinant of
(un)degradability of N in the washout fraction. In
this regard, a correction is used in the Nordic AAT/
PBV-system when calculating the rumen degradability
of protein. In this system, loss of fine particles is
estimated as the difference between total loss from
bags and water-soluble N (Hvelplund and Weisbjerg,
2000). Water-soluble protein is measured as the washing loss over filter paper. The washout fraction corresponds to the true water-soluble fraction and the fine
particle fraction is assumed to degrade in the same
rate as the potentially degradable fraction (Madsen
and Hvelplund, 1993). Madsen et al. (1995) compared
the degradability of protein with the Nordic correction
for the washout fraction and without the correction.
With a rumen outflow rate of 0.05 h 1, the uncorrected degradability ranged from 72.7% to 81.1% for
different concentrate mixtures. For the same feeds, the
corrected degradability markedly differed and ranged
from 56% to 70%. The DVE/OEB-system considers
the losses of the washout fraction as a part of the
potentially undegradable fraction of feeds, using the
correction factor of 1.11, which is taken from the
French PDI System (Tamminga et al., 1994). This
leads to the discussion of the necessity of a more
extended correction for the washout fraction when
calculating undegraded protein, because, to a varying
degree, all protein evaluation systems are based on the
prediction of the amount of undegraded protein reaching the duodenum.
The necessity of a correction is very much dependent upon the degradation rates of FIL, i.e., NS and
TP. Considering the TP measured after 4 h incubation
in vitro (B 4) as percent of TP at 0 h incubation time
(B 0), the potential underestimation of the protein
value of these feeds, calculated as [(NS + TP)/
N feed] B 4(FIL), is 4.5%, 15.0%, 3.6%, 7.6% and
19.9% for SBM, LPM, CGF, CGMS and WBGS,
respectively. The deviation in the prediction of undegradable protein is in agreement with others (Aufrère
et al., 2000). In their study, Aufrère et al. (2000)
227
observed that 7% to 11% of the solubilized protein
of lucerne can escape the rumen, which is in line to
the factor of 1.11 used in the French PDI System to
correct the amount of soluble protein escaping the
rumen (Vérité et al., 1987). In comparison to in vivo
data, the work of Choi et al. (2002a) showed, after
correcting for microbial contamination, that up to 54
g/kg of soluble non-ammonia N (NAN) as proportion
of total dietary NAN was achieved supplementing
lactating cows fed on grass silage with fish meal,
soybean meal or maize gluten meal. In another
work, Choi et al. (2002b) demonstrated that cows
fed grass silage supplemented with rapeseed meal
had higher total dietary NAN flows compared to
cows fed barley, as measured in the liquid phase
entering the omasal canal. The proportion of dietarysoluble NAN entering the duodenum approached 96
g/kg total dietary NAN. For some feeds the soluble
crude protein fraction may contribute 0.10 of the
escape crude protein. The amount observed is in
agreement with the in vitro observations obtained in
the present work, suggesting that there is a fraction of
feed protein that escapes degradation in the rumen
without being considered in the UDP fraction.
5. Conclusion
The procedure reveals the presence of trichloroacetic acid-precipitable protein in the washout fraction
for LPM, SBM and RSM, which is not fully and
rapidly degraded in the rumen as assumed in the
nylon bag method. Fine particles escaping the nylon
mesh have similar degradation rates as the residue left
in the filter. For some feeds, the fraction A may
contribute substantially to the undegradable protein
escaping the rumen. Corrections can be developed
based on the technique presented in this study,
which seems applicable for a wide range of other
feeds.
Acknowledgements
This study was supported by the Dutch Ministry of
Agriculture, Nature and Fisheries and the Dutch Commodity Board of Feed Manufacturers. Our gratitude is
expressed to M. Melin, P. van Wikselaar, V. Hindle,
228
M. Gierus et al. / Livestock Production Science 97 (2005) 219–229
A. Klop and A.A. Mathijssen-Kamman for their support in the laboratory analysis. We thank Prof. K.-H.
Südekum for critical review of the manuscript.
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