Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol81.Issue3-4.Oct1999:

Animal Feed Science and Technology
81 (1999) 279±289

Estimation of net ruminal protein synthesis
from urinary allantoin excretion by bulls
given tropical feeds
M.N. Shema,*, F.D.D. Hovellb, A.E. Kimamboa
a

Department of Animal Science and Production, Sokoine University of Agriculture,
P.O. Box 3004, Chuo Kikuu, Morogoro, Tanzania
b
University of Aberdeen, 581 King Street, Aberdeen, Scotland, AB9 IUD, Scotland, UK
Received 18 June 1998; received in revised form 14 October 1998; accepted 15 June 1999

Abstract
An experiment was carried out to determine net microbial protein supply to ruminants from 15
tropical feeds. The study was done at Lyamungo research institute, on the slopes of Mt.
Kilimanjaro, Tanzania in 1993. Twenty Bos Taurus  Bos Indicus bulls were used in three periods
in a completely randomised design. The initial 19 days of each period were for adaptation by the
bulls to their new environment and diets, followed by 2, 7 and 7 days for total urine collection, spot

urine sampling and digestibility, respectively. The feeds included urea treated and untreated maize
stover (3 feeds), green maize stover (3 feeds), bean straw (2 varieties), cultivated forage (5) and
banana plant residues (2). Both total and spot urine collections were made for estimation of
allantoin. Daily excretion of allantoin ranged from 5.63 to 48.89 mmol/day for banana leaves and
5% urea treated maize stover, respectively. Excretion of allantoin increased with level of dry matter
intake (DMI) for all the feeds. Microbial N supply (MNS) and efficiency of microbial N supply
(EMNS) followed the same trend (4.1±35.5 g N/day and 5.5±17.4 g of N/kg DOMR (digestible
organic matter in the rumen) for banana leaves and 5% urea treated maize stover, respectively.
Correlation coefficients between allantoin excreted (Ae), DMI and BW0.75were r = 0.79 and
r = 0.54, respectively. That between MNS, DMI and BW0.75were r = 0.80 and r = 0.52, respectively,
while that between EMNS, DMI and BW0.75were r = 0.78 and r = 0.54, respectively.
It was concluded that all the tropical feeds tested were poor sources of MNS and EMNS # 1999
Elsevier Science B.V. All rights reserved.
Keywords: Microbial protein; Ruminants; Tropical feeds; Allantoin; Supplementation

*

Corresponding author. Tel.: +255564617; fax: +255564562
E-mail address: shem@suanet.ac.tz (M.N. Shem)
0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 0 8 8 - 7

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1. Introduction
There are a number of methods used to estimate net microbial protein synthesis based
on the use of microbial markers (Sadik et al., 1990). They require the use of postruminally cannulated animals to determine digesta flow which is tedious and has
limitations (Chen, 1989) which might limit their applicability under most tropical
research conditions.
A simpler technique for the determination of net microbial protein synthesis is
based on the determination of total purine derivatives (PD) excreted in the urine of
ruminants (Fujihara et al., 1987; Verbic et al., 1990; IAEA, 1997). The use of allantoin
excretion alone (Vercoe, 1976; Shem, 1993) has also been tried due to the fact that it
comprises 80±85% of excreted PD in the urine of cattle. The preferred method of
collecting urine for PD analysis is the total collection method. However, this method is
difficult under field conditions. As a result, spot urine sampling has been attempted (Chen
et al., 1992a), based on data which indicate that there is relatively constant intestinal flow
of microbial biomass in ruminants throughout the day under normal feeding conditions

(Daniels et al., 1994; Gonda and Linberg, 1994). Where there is an extreme diurnal
variation in digesta flow, corrections for such variation is necessary (Chen et al., 1997).
Prediction equations for microbial N supply developed from PD excretion estimates
depend on a value of 0.85 for digestion and absorption of purines in the small intestines.
The estimate assumes a constant ratio of nucleic acid N to microbial protein N (Bergen
et al., 1982; Zinn and Owens, 1986).
Very few, if any, attempts have been made to determine the microbial protein
contribution of tropical feeds to the nutrition of tropical ruminants under practical
feeding conditions. The aim of this study was therefore, to use the PD excretion
method in the determination of exogenous purine uptake and thus net microbial
protein synthesis in ruminant animals fed on tropical feeds, which could form a basis for
the development of feeding standards to be used in diet formulation for cattle in the
tropics.

2. Materials and methods
An experiment was carried out at Lyamungo Research Institute situated within the
banana±coffee highland zone on the slopes of Mt. Kilimanjaro, Tanzania.
2.1. Animals and their management
A total of 20 Bos taurus  Bos indicus bulls aged between 1 and 2 years with
liveweight ranging from 117 to 209 kg were used in the experiment. The animals were

randomly allocated into five groups of four bulls in a completely randomised change over
design. The experiment was run in three periods each comprising an adaptation period of
19 days and a collection period of 9 days. The animals were housed in individual pens in
a well-ventilated house and were allowed free access to water. Deworming of all animals
was done immediately after confinement using Nilzan (a broad-spectrum anti-helminthic)

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281

according to the maker's instructions. External parasites (mainly ticks) were eliminated
using Ectopor, a pour-on-acaricide, once every 2 weeks.
2.2. Feeds and feeding
Fifteen foodstuffs were used in this experiment. They comprised of maize stover (Zea
mays) from two varieties (Kilima and Malawi) in green form (i.e. residues cut
immediately after fresh maize cobs are harvested) and in the dry form for Malawi only
(i.e. residues left to dry in the fields after dry maize cobs are harvested), 5% urea treated
Malawi dry maize stover (on dry matter), untreated Malawi dry maize stover, Malawi dry
maize stover directly supplemented with 3% urea (on dry matter), green Malawi maize
stover tops and bean straw (Phaseolus vulgaris) from two varieties (Canadian Wonder

and Belabela). Other feeds were banana pseudostems and leaves (Musa spp.) cultivated
forages, which included guatemala grass (Tripsacum fasciculum), setaria grass (Setaria
splendida), napier grass (Pennisetum purpureum) and Rhodes grass (Chloris gayana)
both in its green and hay forms.
During the experiment, feeds were offered to the animals ad libitum,in two equal
amounts at 7.30 and 16.30 h and allowing about 20% excess above appetite. In addition,
each animal was fed 200 g cotton seed cake daily also equally divided between the
morning and afternoon feeds. Cotton seed cake was given to jump-start microbial growth
at the rumen. As the amounts involved were small and given in equal amount to all
animals, it was assumed that the effect of cotton seed cake will not significantly affect the
end results and that the differences in net microbial supply will be mainly due to the feeds
themselves because it was given uniformly across all test feeds. Mineral supplementation
was done according to the recommendation of ARC (1990).
2.3. Urine sample collection and preparation
Total urine collection was made only for 2 days before the commencement of the 7-day
spot sampling period. Urine was collected into 2 l metal containers attached to metal
rods operated manually from individual animals by trained personnel. During the 7-day
spot sample collection period, a minimum of eight samples of urine were also manually
taken (i.e. 1 sample per every 3 h) per day from each animal. Individual urine samples
collected in each period were transferred immediately into containers containing 200 ml

of 10% sulphuric acid in order to prevent bacterial destruction of allantoin in the
urine. The final pH of the urine was maintained below 3. The daily urine samples were
bulked and mixed thoroughly. A representative urine sample (150 ml) from each day's
collection was then taken and diluted with water threefold, filtered through surgical gauze
and stored at ±208C. At the end of the experiment each animal's urine samples were
thawed, bulked and a representative sample (40 ml) taken and stored at ±208C for
allantoin analysis. Total urine output for the 7-day collection period for each animal was
estimated based on the two-day's collection as described above. A correction factor of
plus or minus 3.4 l expressed as ml/kg0.82 were used for the green and dry feeds,
respectively.

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2.4. In vivo digestibility
Digestibility experiment was conducted in a separate study at the end of each period
using the same animal groups, i.e. four animals for each feed. The experimental diets
were offered at 90% of the ad libitum level of intake for 7 days preliminary period after
which total collection of faeces was made for another 7 days. Collected faeces were

thoroughly mixed and bulked and representative samples were taken for the
determination of organic matter (OM) digestibility.
2.5. Chemical analysis
Feed and faeces samples were analysed for DM by drying them in an oven at 608C to a
constant weight for 48 h. Ash content was determined by heating in a muffle furnace at
5508C (AOAC, 1990). N content was analysed by the Kjeldahl method using a semiautomated N analyser. Neutral detergent fibre (NDF) was determined according to Van
Soest et al. (1991).
Allantoin was measured colorimetrically by the method of Young and Conway (1942)
using a normal spectrophotometer (Philips, model PU 8620 UV/VIS/NIR). In this
procedure allantoin was first hydrolysed under weak alkaline condition at 1008C, to
allantoic acid which was also hydrolysed to urea and glyoxylic acid in a weak acid
solution. The glyoxylic acid was reacted with phenylhydrazine hydrochloride to produce
a phenylhydrazone derivative of the acid. The product formed an unstable chromophore
with potassium ferricyanide. The colour was read at 522 nm.
2.6. Calculation of intestinal flow of microbial N
The amount of microbial allantoin absorbed (X mmol/day) corresponding to allantoin
excreted Y (mmol/day) was estimated based on the relationship (Chen et al., 1990):
Y …mmol=day† ˆ 0:85X ‡ 0:385W 0:75
where Y = allantoin excreted and W0.75 = metabolic body weight (kg) of the animal. The
calculation of X from Y based on the above equation was made by means of the Newton's

iteration process. With the assumption that the purine : protein ratio of mixed ruminal
microbes remained constant. The amount of microbial nitrogen (MN g N/day) supply was
calculated using the formulae (Chen, 1989):
MN …g=day† ˆ 70X=…0:830:1161000† ˆ 0:727X
where 0.83 = digestibility coefficient for microbial purines, 70 N content of purines (mg/
mmol), and 0.116 = ratio of purine N to total N in mixed microbial biomass measured
(Chen, 1989).
The `efficiency of microbial nitrogen supply' (EMNS) to denote the microbial N
supplied to the animal per unit of DOMR was calculated using the following formula:
EMNS ˆ

MN …g=day†1000 …g†
DOMR …g†

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where DOMR = DOMI  0.65 (ARC, 1990), DOMR = digestible organic matter
apparently fermented in the rumen and DOMI = digestible organic matter intake.

2.7. Statistical analysis
Analysis of variance of the data was performed with the aid of GENSTAT (Lawes
Agricultural Trust, 1983). Correlation analysis of the relationship between DMI, body
weight, allantoin excretion and efficiency of microbial protein synthesis was also carried
out.
3. Results
The chemical composition of the experimental feeds is given in Table 1. Treating with
or adding urea increased the CP content of maize stover by more than 50% for the two
varieties. Unlike bean straws and dry maize stover, grass forages and banana leaves have
CP values of above 90 g/kg DM. Generally all the feeds were rich in K, its values ranging
from 12 to 36 g/kg DM. The digestibility of DM and OM of maize stover also tended to
increase with urea treatment or supplementation. DM digestibility of the feeds ranged
from 506 g/kgDM in banana leaves to 768 g/kgDM in banana pseudostems (Table 2).
Most parameters (Table 2) showed consistent trend with level of intake and quality of the
feeds.
Daily allantoin excretion is shown in Table 3. It ranged from 5.6 to 48.9 mmol/day for
banana leaves and 5% urea treated maize stover, respectively, and tended to increase with
the level of DMI for all the feeds.
Table 1
Chemical composition of the experimental feeds in terms of dry matter (DM)g/kg (on as fed basis) and in g/kg

DM of crude protein (CP), organic matter (OM) (of the dry sample), neutral detergent fibre (NDF), ash, calcium
(Ca), phosphorus (P) and potassium (P)
Feed
Maize stover
Green Kilima
Green Malawi
5% urea treated Malawi
3% on Malawi
Dry Malawi
Green Malawi maize stover tops
Other feeds
Guatemala grass
Setaria grass
Napier grass
Canadian wonder straw
Belabela bean straw
Rhodes grass (hay)
Rhodes grass (green)
Banana leaves
Banana pseudostems

CP

Om

NDF

Ash

Ca

p

K

73
88
98
89
49
43

926
932
943
941
941
930

773
752
814
826
864
866

74
68
57
59
59
70

4.2
3.3
±
±
1.6
4.3

2.5
1.4
±
±
1.1
2.4

24
15
±
±
13
17

109
90
114
66
48
44
67
127
38

914
918
905
914
925
947
938
937
971

784
788
765
836
864
866
835
659
405

86
82
95
86
75
53
62
63
29

3.0
3.8
3.4
8.7
6.8
3.7
6.6
8.3
9.0

4.0
6.0
4.8
1.0
1.3
2.2
3.8
4.0
0.4

26
36
25
29
18
27
24
30
12

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M.N. Shem et al. / Animal Feed Science and Technology 81 (1999) 279±289

Table 2
Dry matter intake (DMI), organic matter (OM), organic matter digestibility (OMD), digestible organic matter in
the rumen (DOMR) and estimated urine volume excreted by the experimental animals
Feed

DMI
(g/kg0.75)

Maize stover
Green Kilima
Green Malawi
5%urea treated Malawi
3%urea supplemented Malawi
Dry Malawi
Green Malawi maize tops

71.37
82.09
90.11
72.14
70.09
70.78

Other feeds
Canadian wonder bean straw
Belabela bean straw
Guatemala grass
Setaria grass
Napier grass
Green Rhodes grass
Rhodes grass hay
Banana leaves
Banana pseudostems
SEM
Significance

58.44
70.32
70.74
69.82
57.35
86.09
76.22
55.06
44.38
2.56
***

DMD
(g/kg)

OM
(g/kg)

OMD
(g/kg)

649
686
678
604
590
615

926
932
943
941
941
914

671
705
702
631
610
658

651
583
638
685
643
643
612
506
768
3.65
***

918
905
905
925
947
938
930
937
971
1.78
***

651
701
666
602
627
662
633
525
782
0.74
***

DOMI
(kg/day)

DOMR
(kg/day)

Urine volume
(ml/kg0.82)

2.11
2.62
2.62
3.14
1.98
1.66

1.36
1.70
2.03
1.30
1.08
1.37

133.40
113.16
122.36
155.26
166.21
201.93

2.14
1.88
2.40
2.50
1.73
1.90
2.42
2.13
1.15
0.34
**

1.63
1.12
1.39
1.56
1.22
1.57
1.24
0.75
1.05
0.14
**

139.93
123.16
136.88
101.67
97.51
175.59
151.14
191.93
293.05
2.36
***

The microbial N supply ranged from 4.1 for banana leaves to 35.5 g of N/day for 5%
urea treated maize stover. Efficiency of microbial N supply (EMNS) of the feeds ranged
from 5.5 to 17.4 g of N/kg of DOMR for banana leaves and 5% urea treated maize stover,
respectively (Table 3). Correlation coefficients between the different variables were MNS
and DMI (r = 0.80), MNS and BW0.75 (r = 0.52), EMNS and DMI, (r = 0.78), EMNS and
BW0.75, (r = 0.54), allantoin excretion) and DMI, (r = 0.79) and Ae and BW0.75, (r = 0.54)
(formula 1±6) are shown in Table 4.
There was positive relationship between Ae and DMI (Fig. 1) and Ae BW0.75. The
relationship between MNS and DMI (Fig. 2) and MNS and BW0.75 was also positive.
Similarly the relationship between DMI and EMNS (Fig. 3) and BW 0.75 followed the
same trend. In all cases the relationship with DMI was very high while that with body
weight was moderately positive.

4. Discussion
The voluntary intake of urea treated maize stover, green forage and banana
pseudostems was higher compared to that of untreated roughage and banana leaves
due to increased digestibility of treated roughage and lower NDF of the green forage.
Similarly the same trend was observed for apparent digestibility of DM and OM. This
might account for the differences in estimated microbial N supply shown in Table 3. Most
of the excreted allantoin determined in the urine originated from absorbed microbial

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M.N. Shem et al. / Animal Feed Science and Technology 81 (1999) 279±289
Table 3
Net ruminal protein synthesis from urinary allantoina*a excretion
Allantoin excreted
(Ae mmol/day)

W0.75

MN supply
(g N/day)

MN
(g/kg DOMR)

Maize stover
Green Kilima
Green Malawi
5% urea treated Malawi
3% urea supplemented Malawi
Dry Malawi
Green Malawi maize tops

25.5
35.6
48.9
22.9
16.5
24.1

47.5
48.6
52.6
46.3
40.9
50.3

8.5
25.9
35.5
16.7
12.0
17.5

13.5
15.2
17.4
12.9
11.1
13.8

Other feeds
Canadian wonder bean straw
Belabela bean straw
Guatemala grass
Setaria grass
Napier grass
Green Rhodes grass
Rhodes grass hay
Banana leaves
Banana pseudostem
SEM
Significance

22.4
30.8
38.2
17.2
20.4
31.5
26.4
5.6
9.6
1.3
***

53.9
53.9
58.1
44.4
55.8
45.3
47.1
42.8
48.0
0.9
***

16.3
22.4
27.8
12.5
14.8
22.9
19.2
4.1
7.0
1.7
***

11.5
14.1
17.1
11.2
12.0
14.6
14.0
5.5
6.6
1.6
***

Feed

a
These values were corrected to true allantoin excreted by dividing by 0.85 (which assumes that allantoin
comprises 85% of purine derivatives excreted in cattle (Verbic et al., 1990).

Table 4
Relationship between the different variables used in the estimation of microbial nitrogen from different tropical
feed
Variables

Equation

MNS (g/day)
MNS (g/day)
EMNS (g/kgDOMR)
EMNS (g/kg DOMR)
Ae (mmol/day)
Ae (mmol/day)

47.876
43.141
33.275
38.602
48.005
42.942

(7.4652) + 1.1998(0.2480)
(4.3848) + 0.3233(0.1457)
(7.8633) + 2.8401(0.6381)
(4.3360) + 0.8124(0.3518)
(7.6393) + 0.8624(0.1849)
(4.3404) + 0.2417(0.1050)

DMI (g/kg0.75)
BW0.75
DMI (g/kg0.75)
BW0.75
DMI (g/kg0.75)
BW0.75

Correlation

Probability
level

r = 0.802
r = 0.524
r = 0.777
r = 539
r = 0.791
r = 0.538

p < 0.01 [1]
p < 0.05 [2]
p < 0.01 [3]
p < 0.05 [4]
p < 0.01 [5]
p < 0.05 [6]

allantoin as the contribution from endogenous sources is thought to be small (Chen et al.,
1992a). The significant difference (p < 0.05) in the daily allantoin excretion was
primarily due to differences in DMI of the feeds. This is because microbial N estimated
from allantoin excretion is reported to correspond to the amount of microbial biomass
reaching the duodenum rather than that synthesised within the rumen (Chen et al., 1993).
The above reason possibly explains the highly positive correlation coefficient (r = 0.79
p < 0.01) between DMI and Ae. A similar observation was made earlier by Chen et al.
(1992a). Chen et al. (1992b) reported that increased feed intake results in increased

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M.N. Shem et al. / Animal Feed Science and Technology 81 (1999) 279±289

Fig. 1. Relationship between allantoin excreted (Ae) and dry matter intake (DMI).

Fig. 2. Relationships between microbial N supply (g/day) and dry matter intake (DMI).

M.N. Shem et al. / Animal Feed Science and Technology 81 (1999) 279±289

287

Fig. 3. Relationship between efficiency of microbial N supply (EMNS) and dry matter intake (DMI).

microbial N production and hence purine derivative excretion (r = 0.71) in sheep. From
Table 4, it can be seen that feeds which allowed higher DMI intakes also tended to give
significantly (p < 0.05) higher microbial yield with a correlation of (r = 0.80 p < 0.01).
Although BW0.75 was positively correlated with Ae its effect was not as pronounced as
that of DMI.
When the allantoin excretion data was calculated as the supply of microbial N per
kilogram of DOMR (i.e. EMNS), a positive relationship was noted between EMNS and
DMI and BW as shown in Table 4. Efficiency has been shown to be a function of
DMI : BW ratio (not calculated) regardless of the magnitude of the DMI and BW per se
(Chen et al., 1992b). However, the supply of microbial N to the host animal is not solely
determined by the amount of feed consumed. Factors like feed bulk, rumen fill; feed
digestibility and supplementation may increase or decrease net ruminal microbial
synthesis. Values ranging from 14 to 49 g microbial N/kg organic matter apparently
digested in the rumen (DOMR) have been reported (ARC, 1990) and a value of 30 g
microbial N/kg DOMR has been adopted for all diets, whether given to sheep or cattle.
However, ARC (1990) reports a wide variation in values mainly due to the technique used
in determination of the value and that the adopted mean value is not a biological constant
but a mean based on widely varying individual results in literature. As no values on
tropical feeds were included in ARC (1990), it is taken as a guide under the existing
feeding conditions in the study area. Most of the calculated EMNS in this experiment
were on the lower side of the range normally reported in the literature (for temperate
feeds), an indication that the feeds used were poor sources of microbial N on their own
without supplementation. The significance of this data is that, it could form a basis for the

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M.N. Shem et al. / Animal Feed Science and Technology 81 (1999) 279±289

development of feeding standards for use in formulating rations for the different classes
of cattle fed on tropical feeds.
One possible contributing factor to the low levels of microbial N from the feeds could
be the method of urine collection although reasonably consistent volumes were estimated.
Urine volume normally ranges from 5 to 34 kg in cattle (depending on type, size, water
availability and diet) (Paquay et al., 1975). However, this aspect was taken care of by
expressing urine yields as ml/kg0.82. The value of 0.85 (Bergen et al., 1982), for digestion
and absorption of purines in the small intestines might vary with the diet or for other
reasons and may need modifications before generalised prediction equations are derived.
Another possible contributing factor might be that the experimental animals might have
excreted a greater proportion of PD as uric acid than those of Verbic et al. (1990) might.
Other potential sources of error may be from losses of allantoin via non-renal routes,
glomerular filtration rate (GFR) and kidney function.
The method of calculating DOMR from DOMI which assumes the former to be 65%
(ARC, 1990) of the latter has also been questioned (Chen et al., 1992b); thus might have
had an affect on the amount of N yield calculated. Although, uncertainty relating to the
estimation of PD excreted in urine may affect absolute calculated values, it does not
affect the conclusion that all the tropical feeds assessed here were poor sources of
microbial N yield and EMNS. Regardless of the limitations mentioned above, the data is
the first of its kind and could form a basis for future research on this topic. Further
research is needed on microbial yields for a wider range of tropical feeds and feeding
conditions before these data are taken as absolute values. Basic research on the claimed
lower endogenous urinary N excretion by zebu cattle (Bos indicus) than European breeds
(ARC, 1990) needs to be done as the majority of cattle in the tropics belong the zebu
breed.

Acknowledgements
The authors wish to thank Dr. R.N.B. Kay for helpful comments on the manuscript.
Funding from the International Foundation for Science, FAO and NORAD are
acknowledged.

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