Estimation of net ruminal protein synthesis
from urinary allantoin excretion by bulls
given tropical feeds
M.N. Shem
a,*, F.D.D. Hovell
b, A.E. Kimambo
a aDepartment of Animal Science and Production, Sokoine University of Agriculture,P.O. Box 3004, Chuo Kikuu, Morogoro, Tanzania
bUniversity 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. TwentyBos TaurusBos Indicusbulls 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 andr= 0.52, respectively, while that between EMNS, DMI and BW0.75werer= 0.78 andr= 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 81 (1999) 279±289
*Corresponding author. Tel.: +255564617; fax: +255564562
E-mail address: shem@suanet.ac.tz (M.N. Shem)
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 post-ruminally 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 taurusBos 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)
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.
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 semi-automated 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 (Xmmol/day) corresponding to allantoin excretedY (mmol/day) was estimated based on the relationship (Chen et al., 1990):
Y mmol=day 0:85X0:385W0:75
whereY= allantoin excreted andW0.75= metabolic body weight (kg) of the animal. The calculation ofXfromYbased 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 70X= 0:830:1161000 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:
EMNSMN g=day1000 g DOMR g
where DOMR = DOMI0.65 (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 CP Om NDF Ash Ca p K
Maize stover
Green Kilima 73 926 773 74 4.2 2.5 24 Green Malawi 88 932 752 68 3.3 1.4 15 5% urea treated Malawi 98 943 814 57 ± ± ±
3% on Malawi 89 941 826 59 ± ± ±
Dry Malawi 49 941 864 59 1.6 1.1 13
Green Malawi maize stover tops 43 930 866 70 4.3 2.4 17
Other feeds
Guatemala grass 109 914 784 86 3.0 4.0 26 Setaria grass 90 918 788 82 3.8 6.0 36 Napier grass 114 905 765 95 3.4 4.8 25 Canadian wonder straw 66 914 836 86 8.7 1.0 29 Belabela bean straw 48 925 864 75 6.8 1.3 18 Rhodes grass (hay) 44 947 866 53 3.7 2.2 27 Rhodes grass (green) 67 938 835 62 6.6 3.8 24 Banana leaves 127 937 659 63 8.3 4.0 30 Banana pseudostems 38 971 405 29 9.0 0.4 12
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) andAeand 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 0.75followed 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
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
Green Kilima 71.37 649 926 671 2.11 1.36 133.40 Green Malawi 82.09 686 932 705 2.62 1.70 113.16 5%urea treated Malawi 90.11 678 943 702 2.62 2.03 122.36 3%urea supplemented Malawi 72.14 604 941 631 3.14 1.30 155.26 Dry Malawi 70.09 590 941 610 1.98 1.08 166.21 Green Malawi maize tops 70.78 615 914 658 1.66 1.37 201.93
Other feeds
Canadian wonder bean straw 58.44 651 918 651 2.14 1.63 139.93 Belabela bean straw 70.32 583 905 701 1.88 1.12 123.16 Guatemala grass 70.74 638 905 666 2.40 1.39 136.88 Setaria grass 69.82 685 925 602 2.50 1.56 101.67 Napier grass 57.35 643 947 627 1.73 1.22 97.51 Green Rhodes grass 86.09 643 938 662 1.90 1.57 175.59 Rhodes grass hay 76.22 612 930 633 2.42 1.24 151.14 Banana leaves 55.06 506 937 525 2.13 0.75 191.93 Banana pseudostems 44.38 768 971 782 1.15 1.05 293.05 SEM 2.56 3.65 1.78 0.74 0.34 0.14 2.36 Significance *** *** *** *** ** ** ***
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
Table 3
Net ruminal protein synthesis from urinary allantoina*aexcretion
Feed Allantoin excreted
Green Kilima 25.5 47.5 8.5 13.5
Green Malawi 35.6 48.6 25.9 15.2
5% urea treated Malawi 48.9 52.6 35.5 17.4 3% urea supplemented Malawi 22.9 46.3 16.7 12.9
Dry Malawi 16.5 40.9 12.0 11.1
Green Malawi maize tops 24.1 50.3 17.5 13.8
Other feeds
Canadian wonder bean straw 22.4 53.9 16.3 11.5 Belabela bean straw 30.8 53.9 22.4 14.1 Guatemala grass 38.2 58.1 27.8 17.1 Setaria grass 17.2 44.4 12.5 11.2
Napier grass 20.4 55.8 14.8 12.0
Green Rhodes grass 31.5 45.3 22.9 14.6 Rhodes grass hay 26.4 47.1 19.2 14.0
Banana leaves 5.6 42.8 4.1 5.5
Banana pseudostem 9.6 48.0 7.0 6.6
SEM 1.3 0.9 1.7 1.6
Significance *** *** *** ***
aThese 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
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).
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= 0.80p< 0.01). Although BW0.75was positively correlated withAeits 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
Fig. 3. Relationship between efficiency of microbial N supply (EMNS) and dry matter intake (DMI).
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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