Hof, G.

Kouwenberg, W.J.A.

Tamminga, S.

English

Wageningen Yield 

Netherlands Journal of Agricultural Science 38 (1990) 719-724 SYNOPSIS 
The effect of washing procedure on the estimation 
of the in situ disappearance of amino acids from 
feed protein 
G. HOF1, W. J. A. KOUWENBERG2 & S. TAMMINGA' 
1 Department of Animal Nutrition, Wageningen Agricultural University, Haagsteeg 4, 
NL 6708 PM Wageningen, Netherlands 
2 Present adress: Sondervick 99, NL 5505 NC Veldhoven, Netherlands 
Received 7 September 1989; accepted 26 June 1990 
Abstract 
In a small scale experiment the in-situ disappearance of amino acids and microbial contami­
nation of the incubation residues were studied to investigate the influence of rumen incuba­
tion on the amino acid profile. Wilted grass silage, soybean expeller and hydrolysed feather-
meal were used for the purpose. Incubation time ranged from zero till 48 h. Three washing 
procedures were compared: a) standard procedure, b) standard washing, followed by soaking 
for 16 h in ND at 70 °C, followed by standard washing and c) a comparable procedure but 
using a ND solution at 25 °C. The amino acid profile appeared to be almost exclusively deter­
mined by amino acid content in the original feed. Amino acids disappeared at a faster rate 
than non-amino acid N. Microbial contamination was only observed in the grass silage and 
its residues after incubation. Non of the washing procedures removed effectively the contami­
nation. 
Keywords: protein degradation, in situ measurement, microbial contamination, washing 
procedure 
Introduction 
The response of ruminants to feed protein basically depends on the amount and na­
ture of amino acids absorbed from the small intestine. This amount is usually esti­
mated from the fraction of dietary protein escaping microbial degradation in the ru­
men, microbial protein synthesis in the rumen and the digestibility of both fractions. 
At present such predictions presume no difference in amino acid composition be­
tween the protein originally present in the feed and that escaping microbial degrada­
tion. Hence, the amino acid profile is not predicted correctly, if protein components 
differ in amino acid composition and rumen degradability. 
The nylon bag technique is a widely accepted method for estimating the propor­
tion of protein escaping rumen degradation (Mehrez & Orskov, 1977). Sometimes 
the feed residues after incubation are contaminated with microbial protein, which 
results in an underestimation of protein degradation (Nocek, 1987). The prediction 
719 
G. HOF, W. J. A. KOUWENBERG AND S. TAMMINGA 
of the release of individual amino acids from feed protein with the in situ technique 
may be even more biased by microbial contamination when amino acid profile of 
the feed differs considerably from that of microbial protein. 
Van Soest (1983) suggests that microbial contamination in digestive residues can 
be eliminated by washing with Neutral Detergent (ND). Boiling the residue in ND 
may not only remove microbial contamination, but also part of the insoluble feed 
protein. Therefore a milder treatment with ND at room temperature may be prefera­
bly. 
In a small scale experiment the in situ disappearance of amino acids and microbial 
contamination of the incubation residues were studied in three feeds. The objectives 
of the experiment were: 
— to estimate possible differences between the amino acid profile of residues after 
rumen incubation and of the original feed protein, 
— to quantify microbial contamination in feed residue, 
— to evaluate washing procedures on their effectiveness to eliminate microbial con­
tamination. 
Experimental procedures 
Amino acid disappearance from nylon bags, suspended in the rumen of rumen can-
nulated dairy cows and containing wilted grass silage, soybean expeller or hydro-
lyzed feathermeal was measured according to a standardized procedure (Tamminga 
et al., 1989). Wilted grass silage was chosen as an example of feeds with a moderate 
protein content, which may partly be protected from degradation in the rumen be­
cause of its inclusion in cell walls. Feathermeal, on the other hand, is an example 
of feedstuffs with virtually no cell walls. Soybean meal combines a fairly high pro­
tein content with a moderate level of cell walls. Fresh grass silage was chopped to 
an average particle size of approximately 1 cm, the other feeds were ground over 
a screen with 1 mm pore size. Rumen incubation times of soybean meal were 0, 3, 
6 and 12 hours, those of grass silage and feathermeal 0, 6, 24 and 48 hours. 
Three washing procedures were compared on their effect on amino acid content 
in the residues and on their effectiveness in the removal of microbes from the incu­
bation residues: 
a. standard procedure, using a domestic washing machine with cold water and no 
spinning (Tamminga et al., 1989), 
b. washing according to (a), followed by soaking for 16 h in ND at 70 °C (Geuring 
& van Soest, 1979), followed by standard washing, 
c. as (b), but ND temperature at 25 °C. 
Feed and incubation residues were analysed for dry matter (ISO 6496), nitrogen 
(ISO 5983), ash (ISO 5989), amino acids and diaminopimelic acid (DAPA) content 
(Stadler & Tanner, 1984). 
The data were statistically analysed (SAS, 1985) according to the following 
model: 
y- i ju  =  u  + fi  +  Wj + t k  + two-way in teract ions  + e i j k t  
720 Netherlands Journal of Agricultural Science 38 (1990) 
IN SITU DISAPPEARANCE OF AMINO ACIDS FROM FEED PROTEIN 
where: 
yiJk, = characteristic of the ijkl'h residue 
u = overall mean 
fi = effect of the /'th feed 
Wj = effect of the fh washing procedure 
tk = effect of the A?h incubation time 
e l j k l  = error term 
Results and discussion 
Feathermeal had the highest proportion of amino acids per 16 gram N and grass 
silage the lowest (Table 1). The amino acid content in the three feedstuffs seem to 
be relatively low, however, histidine, phenylalanine, tyrosine and tryptophane were 
not measured. Corrected for these missing amino acids by using values from feed 
tables (CVB, 1988) the amino acids in soybean meal and feathermeal represented 
96 and 90 % of the expected values, respectively. 
Individual amino acids disappeared at a slightly different rate, especially in soy­
bean meal (Fig. 1). However, the differences were small and usually within the limits 
of analytical accuracy. In both concentrates the rate of amino acid disappearance 
was not much different from that of total protein. The disappearance of amino 
acids from grass silage protein was significantly higher than that of total protein af­
ter 24 hours incubation onwards. Table 1 shows for grass silage a lower proportion 
of amino acid N as compared to the other two feeds. The insoluble non-amino acid 
N in forage is assumed to be associated with the lignocellulosic complex (van Soest, 
1983) and therefore released at a slower rate. 
DAPA was only found in grass silage, which contained 0.349 g DAPA N per 100 
g N in the original feed (Fig. 1). Assuming 0.6 g DAPA N per 100 g microbial pro­
tein N (Nikolic & Jovanovic, 1973), about 58 °7o of the N in the silage would be of 
microbial origin. This is considerably more than usually found in grass silages 
(Rooke et al., 1984; Ullrich, 1984). Intensive lysis during the ensiling proces, or an 
inseparable co-eluting compound present in the silage (Crooker et al., 1987) may be 
responsible for this. 
Table 1. Average amino acid in grams/16 grams N in feed and after incubation (standard washing proce­
dure a). 
Grass silage Soybean meal Hydrolysed feathermeal 
In feed 67.9 84.5 86.3 
After incubation 
0 h 70.2 81.6 87.4 
3 h - 82.2 -
6 h 71.6 82.9 90.2 
12 h - 82.4 -
24 h 66.9 - 90.6 
48 h 64.9 - 86.8 
Netherlands Journal of Agricultural Science 38 (1990) 721 
16 
14 
12 
10 
8 
6 
4 
2 
0 
22 
20 
18 
16 
14 
12 
10 
8 
6 
4 
2 
0 
16 
14 
12 
10 
8 
6 
4 
2 
0 
l. , 
W. J. A. KOUWENBERG AND S. TAMMINGA 
GRASS SILAGE 
II 
SOYBEAN MEAL 
[fel 
HYDROLYSED FEATHERMEAL 
1 
\LA ARG ASP DAPA GLU GLY ILE LEU LYS M+C PRO SER THR VAL 
rreatm I I 
feed 
T////À PFml 
0  h r  3 / 6  h r  6 / 2 4  h r  1 2 / 4 8  h r  
mino acid pattern in nylon bag residue. 
Netherlands Journal of Agricultural Science 38 (1990) 
IN SITU DISAPPEARANCE OF AMINO ACIDS FROM FEED PROTEIN 
Table 2. Least square means of DAPA N (in % of total amino acid N) after incubation. 
Incubation time (h) Mean (%) 
0 
6 
24 
48 
0.352a l 
0.364a 
0.420ab 
0.448b 
' Significant differences are indicated by different letters. 
Microbial contamination of the feed residue, based on DAPA concentration in 
the residues after incubation, seemed to become relatively more important with time 
of incubation, as it disappeared significantly slower from the nylon bags than the 
other amino acids (Table 2). Microbes were firmly attached to the feed because 
DAPA could only partly be removed by washing. After standard washing 55 and 
14 % of the original DAPA was still present in the fresh sample and the residue after 
48 h incubation, respectively. For the washing procedures with ND at 25 and 70 °C 
these values were 32, 9 and 40,8 %, respectively. 
Soaking in ND at 25 °C and at 70 °C increased on average protein solubility in 
the original samples to 42.0 and 64.2 % as compared to 16.2 % after washing with 
water only. Hence, ND not only removed microbial contamination, it also solubi-
lized part of the insoluble feed protein. This effect was significantly higher in soybe­
an meal than in feathermeal. The effect on grass silage was intermediate. The rate 
(£d) of protein degradation and amino acid release was not influenced by washing 
procedure. 
Conclusions 
— The profile of amino acid residue is almost exclusively determined by the original 
feed. Differences in rate of amino acid disappearance from nylon bags incubated 
in the rumen seem to exist, but are only of minor importance. 
— Amino acids disappeared at a faster rate from the sample than non-amino acid 
N, particularly in grass silage. 
— The problem of microbial contamination appears to be restricted to feeds rich 
in cell wall constituents, because it was only observed in grass silage and not in soy­
bean meal and feathermeal. 
— DAPA-N in grass silage and its residues after incubation was not eliminated by 
washing in ND. ND at 25 °C or 70 °C not only reduced microbial contamination, 
but also solubilized part of the insoluble protein, especially in soybean meal. 
Acknowledgement 
Amino acids and DAPA content were determined by Degussa AG. 
Netherlands Journal of Agricultural Science 38 (1990) 723 
G. HOF, W. J. A. KOUWENBERG AND S. TAMMINGA 
References 
CVB, 1988. Feedstuff table (In Dutch). Centraal Veevoederbureau in Nederland, Lelystad. 
Crooker, B. A., J. H. Clark, R. D. Shanks & G. J. Fahey Jr., 1987. Effect of ruminai exposure on the 
amino acid profile of feeds. Canadian Journal of Animal Science 67: 1143-1148. 
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Mehrez, A. Z. & E. R. Orskov, 1977. A study of the artificial fibre bag technique for determining the 
digestibility of feeds in the rumen. Journal of Agricultural Science (Cambridge) 88: 645-650. 
Nicolic, A. J. & M. Jovanovic, 1973. Preliminary study on the use of different methods for determining 
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bluwgnl). 
724 Netherlands Journal of Agricultural Science 38 (1990) 


The effect of washing procedure on the estimation of the in situ disappearance of amino acids from feed protein

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The effect of washing procedure on the estimation of the in situ disappearance of amino acids from feed protein

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