Animal Feed Science and Technology
92 (2001) 203±214
Urinary excretion of duodenal purine derivatives
in Kedah-Kelantan cattle
O. Pimpaa, J.B. Lianga,*, Z.A. Jelana, N. Abdullahb
a
Department of Animal Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
b
Department of Biochemistry, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Received 4 January 2000; received in revised form 25 April 2001; accepted 11 May 2001
Abstract
Three male Kedah-Kelantan (KK) cattle each ®tted with a ruminal and a T-shaped duodenal
cannulae, with an initial body weight of 178:3 5:78 kg were used to study the recovery rate of
urinary purine derivatives (PD) after duodenal infusion of incremental amounts of purine bases
(PB). During the experiment, the cattle were fed at a maintenance energy level with a diet
containing 40% oil palm frond and 60% concentrates. Basal purine ¯ows into the duodenum from
the maintenance diet were estimated. Purine bases in the form of adenosine (46%) and guanosine
(54%) were infused into the duodenum in four incremental rates equivalent to 10, 15, 30 and
45 mmol purine per day. Urinary allantoin, the principal PD was linearly correlated with PB input,
while the contributions of other PD were not affected by treatments. The relationship between daily
urinary PD (allantoin, uric acid, hypoxanthine and xanthine) excretion (mmol per day) and
duodenal PB ¯ow (mmol per day) was Y ˆ 0:847X ‡ 7:146 (r 2 ˆ 0:50, P < 0:001), suggesting that
0.85 of the supplied exogenous PB were excreted in urine, with an endogenous excretion of
7.15 mmol per day. Urinary PD excretion rates of zebu cattle are similar to those of European cattle.
# 2001 Elsevier Science B.V. All rights reserved.
Keywords: Zebu cattle; Urinary purine derivatives; Rumen microbial protein
1. Introduction
Microbial protein in digesta ¯owing from the forestomachs to the small intestine often
supplies the majority of amino acids required by ruminants. Topps and Elliott (1965) were
among the earliest investigators to suggest that urinary allantoin and uric acid excretion
rates re¯ect the amount of microbial protein ¯owing into the small intestine. Later,
*
Corresponding author. Tel.: ‡60-3-948-6101; fax: ‡60-3-943-2954.
E-mail address: [email protected] (J.B. Liang).
0377-8401/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 1 ) 0 0 2 5 9 - 0
204
O. Pimpa et al. / Animal Feed Science and Technology 92 (2001) 203±214
McAllan and Smith (1973), Rys et al. (1975), McAllan (1980), Razzaque et al. (1981) and
Schelling et al. (1982) con®rmed that urinary purine derivatives (PD) can be an accurate
index of rumen microbial protein ¯owing into the small intestine.
Verbic et al. (1990) infused several levels of microbial nucleic acids into the abomasum
of steers totally nourished by intra-gastric infusion and reported a recovery rate of 0.77 in
the form of urinary allantoin and uric acid. Based on their ®ndings, these authors further
developed prediction equations for rumen microbial ¯ow into the small intestine for steers.
Similarly, relationships between exogenous abomasally infused purines and rates of
urinary purine excretion for European sheep were established in several laboratories
(Antoniewicz and Pisulewski, 1982; Fujihara et al., 1987; Chen et al., 1990; Balcells et al.,
1991; Perez et al., 1996). However relationships established for European animals may not
be applicable for tropical zebu cattle and water buffaloes (Vercoe, 1976; Liang et al., 1994).
It is important that equations developed with European cattle be validated with and for
tropical zebu cattle.
This study reports endogenous excretion, and renal excretion rates, of PD in Malaysian
zebu Kedah-Kelantan (KK) cattle duodenally infused with incremental levels of exogenous
purine. The objective was to determine if a relationship between urinary excretion of PD
and purine input to the small intestine of zebu cattle occurs.
2. Materials and methods
2.1. Animals and management
Three male KK cattle with an initial body weight of 178:3 5:78 kg were ®tted with a
ruminal and a polyvinyl choride (PVC) T-shaped duodenal cannula, 8 cm from the
pylorus 1 month before the experiment began. Animals were kept in individual
metabolism crates and fed at maintenance energy levels of 1% dry matter (DM) of
BW. The experimental diet consisted of 40% oil palm frond and 60% concentrate (maize
grain 24%, soy bean meal 29.5%, cassava chip 29.5% and mineral premix 1.7% on DM
basis) mixed and pelleted together with a calculated energy content of 8.4 MJ ME/kg DM
containing 12.9% CP, 52.0% NDF, 25.7% ADF and 7.7% ash on a DM basis (Table 1).
The daily feed was offered in two equal portions at 09.00 and 16.00 h. Clean water was
Table 1
Dry matter (DM) and major chemical components of dietary ingredients
Oil palm frond (OPF)
Concentratea
Dietb
Dry matter
(g/kg)
Crude protein
(g/kg DM)
Neutral detergent
fiber (g/kg DM)
Acid detergent
fiber (g/kg DM)
Ash
(g/kg DM)
954
964
959
54.9
168.3
128.8
846.6
419.0
519.9
567.6
111.0
257.3
67.5
47.2
76.5
a
Consisted of maize grain 24%, soy bean meal 29.5%, cassava chip 29.5% and mineral premix 1.7% on DM
basis.
b
Consisted of 40% oil palm frond and 60% concentrate.
O. Pimpa et al. / Animal Feed Science and Technology 92 (2001) 203±214
205
Fig. 1. Schedule of experimental procedure.
available at all times. Body weights of the animals were recorded immediately before and
after the experiment.
2.2. Experimental design
The experiment was conducted over a period of 45 days, which consisted of 7 days of
adaptation, 7 days for ®rst measurement of the ``basal'' purine level in duodenal digesta, 24
days (four successive 6-day periods) of purine bases (PB) infusion in a 3 4 incomplete
Latin Square design, and 7 days of second measurement of basal purine in duodenal digesta
(Fig. 1). The second measurement of the basal purine level in duodenal digesta was
repeated immediately after PB infusion because possible technical errors were detected
during the ®rst measurement. Results of the ®rst measurement were not used.
2.3. Measurement of basal purine in duodenal digesta
Basal purine levels in duodenal digesta were estimated using the dual marker technique
(Faichney, 1975). Yb acetate (100 mg Yb/kg DM) and Co-EDTA (78.4 mg Co/kg DM)
solutions were continuously infused into the rumen of each animal through two independent lines by means of a multi-channel peristaltic pump (Gilson, model 312) for 7 days.
Duodenal digesta samples (150±200 ml each) were collected during the last 2 days of
marker infusion at approximately 6 h intervals for 48 h. Digesta samples were immediately
stored at 208C and later pooled on an individual animal basis. Each pooled sample was
homogenized (Heidolph, Model DIAX 600, Germany) at 8000 rpm for 2 min and divided
equally into two portions. One portion was used as a representative of pooled whole
duodenal digesta, the other portion was centrifuged at 1000 g for 5 min, and the pellet
obtained represented the pooled particulate duodenal digesta. Composite samples of whole
duodenal digesta and particulate matter (Faichney, 1975) were freeze-dried and the
samples were ashed and digested for Co and Yb analysis.
2.4. Purine bases (PB) infusion
The infusion solutions (adenine 46.4%, guanine 53.6%) containing the equivalent of 10
(L10), 15 (L15), 30 (L30) and 45 (L45) mmol purine, respectively, were prepared the night
206
O. Pimpa et al. / Animal Feed Science and Technology 92 (2001) 203±214
prior to infusion by dissolving the required quantities of Adenosine (Sigma1 FW 283.2)
and Guanosine (Duechfa1 FW 267.2) in 200 ml of 0.1N NaOH. The solution was later
diluted to 600 ml using distilled water and its pH adjusted to 8 with concentrated HCl. The
prepared solutions were stored at 48C for infusion the following morning.
A 3 4 incomplete Latin Square design was employed. Each of the three cattle was
randomly allocated to one of the four infusion rates per period in a manner that all animals
completed the four treatments by the end of the fourth period. Each period consisted of 6
days of infusion (i.e. 1 day of adaptation and 5 days of sampling). Infusion started at about
08.30 h with the infusion solutions being continuously stirred mechanically and delivered
into the duodenal cannulae via a vinyl tube (3 m length with an internal diameter of about
4 mm) using the same peristaltic pump for the infusion of Yb and Co markers described
previously.
2.5. Urine and blood sampling and analyses
Urine and blood samples were collected during the last 5 days of each PB infusion
period. Urine of individual animals was collected in 200 ml of 20% H2SO4 to keep the ®nal
pH of the urine lower than 3. After recording the weight, urine was diluted to 20 kg with tap
water and mixed thoroughly. Duplicate urine samples of 50 ml were taken and stored at
208C for later analysis.
Blood was sampled from jugular veins during the last 5 days of each period at about
14.00 h in heparinized tubes, which were later centrifuged at 2200 g for 20 min. The
plasma obtained was stored at 208C for later analysis.
2.6. Analytical methods
Urinary and plasma PD and creatinine were determined using HPLC analysis which
consisted of a multi-solvent delivery system (Waters model 600 E, USA), an injector
(WISTTM model 712), a multi-wavelength detector (model 490E; set to 205 nm) and a
double 4:6 mm 250 mm C-18 reverse-phase column (Spherisorb1) following the procedure of Balcells et al. (1992). Purine derivatives were quanti®ed by peak integration
using Waters HPLC system controller software Maxima 820.
Liquid digesta were diluted by 0.1 M HCl 1:5 (liquid digesta:HCl, v/v) (Okine et al.,
1989) for determination of Co and Yb concentration in the liquid phase. Whole and
particulate duodenal digesta samples were freeze-dried and ashed in a muf¯e furnace at
6008C for 8 h. They were then digested in 3 ml phosphoric acid±manganese sulfate
solution (30 ml of 10%, w/v, potassium bromate), on a preheated hot plate (150±2008C)
until there were no more bubbles and a purple color appeared. The digested samples were
cooled and transferred completely by washing with deionized water. The volume was made
up to 50 ml with deionized water, mixed thoroughly, and left to stand overnight for the
suspended materials to settle. Both Co and Yb concentrations were determined using 240.7
and 398.8 nm wavelengths, respectively, by atomic absorption spectroscopy (Varian1
SpectAA-400, Australia) procedure. Whole and particle digesta was freeze-dried and
ground through a 2 mm screen and analyzed for DM and purine contents. Purine
concentrations in adenine, guanine and duodenal digesta samples were determined by
O. Pimpa et al. / Animal Feed Science and Technology 92 (2001) 203±214
207
the same HPLC technique, after acid hydrolysis with 2 ml of 2 M perchloric acid (PCA) at
1008C for 1 h, adding 0.75 mmol of allopurinol and neutralizing immediately with 4.5 M
KOH (Martin Orue et al., 1995). The amount of purine base ¯owing into the duodenum for
each animal was calculated by reference to Co±EDTA and Yb±Ac as liquid and particulate
markers, respectively (Faichney, 1980).
2.7. Calculation of glomerular filtration rate (GFR), tubular load (TL),
and reabsorption (RB)
The glomerular ®ltration rate (GFR, liter per day) was calculated from the relationship
between urinary creatinine excretion rate (A (mmol per day)) and the plasma creatinine
concentration (B (mmol/l)) as GFR ˆ A=B.
Tubular load (TL) of allantoin (mmol per day) was estimated as GFR (liter per
day† plasma allantoin concentration (mmol/l) and reabsorption (RB, mmol per day)
of allantoin was estimated as TL of allantoin Ð allantoin excretion in urine (mmol per day)
following the procedure described in IAEA-TECDOC-945 (1997). The same calculation
procedures were used for estimations of TL and RB of uric acid. The above calculations
were done on a daily basis for each animal and later averaged within animal for each
period.
2.8. Data analyses
Urinary and plasma PD, GFR, TL and RB of PD were compared among purine
infusion levels using the general linear models procedures of the SAS (Statistical
Analysis Systems Institute Inc., 1988). Relationships between the excretion of urinary
PD (mmol per day) and total PB supplied (mmol per day) (i.e. sum of infusion doses and
basal purine in duodenal digesta) was established using the linear regression of SAS
(1988).
3. Results
3.1. Conditions of cattle
The three cattle remained in good health throughout the experiment. There were no
substantive changes in their body weights, indicating that maintenance energy intake was
maintained.
3.2. Basal purine in duodenal digesta
Daily digesta ¯ow did not differ among cattle (Table 2). Purine base (PB) concentrations
in the fresh digesta did not differ among cattle and since basal digesta purine ¯ow also did
not differ among animals, the mean value of 9.8 mmol per day was used as the quantity of
endogenous purine ¯owing into the duodenum. This value was added to the respective
exogenous purines infused to the duodenal to give the ®nal purine doses of 19.8, 24.8, 39.8
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O. Pimpa et al. / Animal Feed Science and Technology 92 (2001) 203±214
Table 2
Estimated daily flow rate of digesta, purine bases (PB) concentration in digesta and daily PB flowa
Animal
S.E.M.
1
2
3
Digesta flow rate (kg per day)
Total
Dry matter
29.21
1.12
26.64
0.94
32.35
1.13
1.533
0.044
PB in digesta (mmol/kg)
Total
Dry matter
0.35
10.83
0.38
11.20
0.27
11.96
0.019
0.906
Purine base flow (mmol per day)
10.23
10.31
8.81
0.613
a
There were no differences (P > 0:01) among cattle for any parameter.
and 54.8 mmol per day for the four treatments. These values were used to examine
relationships with PD excretions (Fig. 2).
3.3. Purine derivatives recovery
Allantoin was the principal PD detected in urine, followed by uric acid. Hypoxanthine
and xanthine concentrations were negligible. The allantoin to total PD ratio increased
linearly with increased levels of exogenous purine input. The response of uric acid to
increased purine base infusion was to decline at a decreasing rate with the minimum
occurrence with 30 mmol per day exogenous purine base (Table 3). Allantoin:uric acid
ratios in the urine increased linearly with increasing level of exogenous PB infusion. Daily
total PD recovery increased linearly with increasing levels of exogenous purine input.
Daily creatinine and urinary nitrogen excretions were not affected by treatments, with
mean values of 768 mmol/kgW0.75 and 283 mg/kgW0.75, respectively.
Fig. 2. Urinary excretion of PD in relation to purine flows to duodenal in KK cattle given a duodenal infusion
of PB.
O. Pimpa et al. / Animal Feed Science and Technology 92 (2001) 203±214
209
Table 3
Daily excretion of urinary PD, creatinine, urine nitrogen and recovery rates as derivatives in urine in the different
treatments
Level of exogenous purine (mmol per day)
Allantoin (mmol/kgW0.75)
Uric acid (mmol/kgW0.75)
Hypoxanthine (mmol/kgW0.75)
Xanthine (mmol/kgW0.75)
Total PD (mmol/kgW0.75)
Recoveryb (%)
Allantoin (%)
Uric acid (%)
Allantoin ‡ uric acid (%)
Allantoin:uric acid
Urine nitrogen (mg/kgW0.75)
Creatinine (mmol/kgW0.75)
10
15
30
45
348
100.4
7.12
6.02
462
71.5
75.4
22.1
96.8
3.47
274
781
640
105.9
25.76
15.27
787
91.0
80.9
13.4
93.7
6.04
292
789
725
70.0
21.32
7.35
848
84.4
85.3
8.3
93.6
10.35
325
731
1163
113.0
13.41
12.43
1302
87.4
89.6
8.8
98.4
10.29
242
772
S.E.M.
44.0
7.18
2.36
6.89
46.2
5.11
1.62
1.39
0.64
0.92
13.8
44.3
Significance of
treatment effects
L
Q
***
NSa
NS
NS
NS
NS
***
NS
***
***
NS
***
NS
NS
**
NS
NS
NS
NS
**
*
NS
NS
NS
a
NS: not significant, P > 0:05.
Calculated as: (total excretion
*
P < 0:05.
**
P < 0:01.
***
P < 0:001.
b
7:15)/(purine infused ‡ 9:77) (mmol per day).
3.4. Purine derivatives excretion in relation to purine supply
Total PD excretion (mmol per day) was linearly correlated with purine supplied (mmol
per day) as described by the equation Y ˆ 0:847X ‡ 7:146 (r 2 ˆ 0:50, P < 0:001) (Fig. 2).
The slope of the above equation indicates an excretion rate of 0.85 as urinary PD in KK
cattle given duodenal infusion of purine bases, while the intercept suggests an endogenous
urinary excretion of 7.15 mmol per day.
3.5. The concentration of PD in plasma
As in urine samples, the concentration of hypoxanthine and xanthine in plasma samples
were negligible and in many cases non-detectable by HPLC and, thus, their contributions
are excluded. There was a linear increase in plasma allantoin concentration with increased
rate of purine infusion, although this does not appear entirely consistent with the data
(Table 4).
3.6. GFR, TL and RB
Glomerular ®ltration rate (GFR) did not differ among treatments and averaged 271 l per
day (Table 4). Similarly, TL of allantoin, uric acid and total (i.e. allantoin plus uric acid)
also did not differ among treatments. However, RB of allantoin and total PD decreased
linearly as the supply of exogenous purines increased (Table 4).
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O. Pimpa et al. / Animal Feed Science and Technology 92 (2001) 203±214
Table 4
PD and creatinine concentrations in plasma, glomerular filtration rate (GFR), tubular load (TL) and net
reabsorption (RB) of purine in KK cattle in different treatments
Level of exogenous purine (mmol per day)
S.E.M.
10
15
30
45
Plasma (mmol/l)
Allantoin
Uric acid
Total PD
Creatinine
190.4
25.8
216.8
134.1
278.3
38.4
317.5
137.8
273.2
28.3
302.8
138.8
290.4
34.0
324.7
142.8
10.95
2.62
11.63
3.91
GFR (liter per day)
280
273
258
264
17.2
Significance of
treatment effects
L
Q
**
NS
NSa
NS
NS
NS
NS
NS
NS
**
TL (mmol per day)
Allantoin
Uric acid
Total
55.1
7.4
62.6
73.6
8.8
82.6
67.5
6.7
74.6
63.8
8.2
72.0
4.50
0.57
4.76
NS
NS
NS
NS
NS
NS
RB (mmol per day)
Allantoin
Uric acid
Total
44.5
4.6
49.0
49.5
4.7
54.6
28.7
4.1
33.4
19.5
5.9
26.4
4.07
0.63
4.27
*
NS
NS
NS
NS
*
a
NS: not significant, P > 0:05.
P < 0:05.
**
P < 0:01.
*
4. Discussion
4.1. Basal purine in duodenal digesta
Purine base contents of whole duodenal digesta estimated in this study were within
ranges reported for sheep (Perez et al., 1998) using similar marker techniques. Verbic et al.
(1990) used steers totally nourished by intragastric nutrition, arguing that this method
enabled precise control of nutrient input and, since there was no microbial fermentation in
the rumen of the animals, more accurate measurements of treatment effects. However,
Orellana Boero et al. (1999) suggested that the procedure of feeding the animals with a
maintenance diet better simulates normal feeding conditions.
4.2. Purine derivatives recovery
That allantoin excretion comprised the major proportion of the total urinary PD agrees
with values reported by Verbic et al. (1990) for European steers receiving exogenous
microbial nucleic acid infusions as well as values reported for zebu KK cattle fed
at maintenance level (Liang et al., 1994). However, our values were lower than the
89±97% reported by Giesecke et al. (1994) and Vagnoni et al. (1997). The negligible
amounts of hypoxanthine and xanthine recorded in the present study con®rm the high
O. Pimpa et al. / Animal Feed Science and Technology 92 (2001) 203±214
211
reported activity of xanthine oxidase in most tissues and blood of cattle (Al-Khalidi and
Chaglassian, 1965).
The linear increase in the proportion of allantoin to total PD with increased rate of
exogenous purine inputs was compensated for by the higher uric acid excretion. This ratios
of allantoin plus uric acid to total PD showed less variation among the treatments, and are
in agreement with those reported for steers by Verbic et al. (1990) and Chen et al. (1992).
4.3. The relationship between total PD excretion and PB absorption
Several groups (Giesecke et al., 1984; Fujihara et al., 1987; Chen et al., 1990; Verbic
et al., 1990) used a linear model to describe the relationship between PD excretion and
purine absorption. Such a model assumes that endogenous excretion and the incremental
recovery are constant. Using a similar linear model, the relationship between PD excretion
(mmol per day) and PB absorption (mmol per day) was estimated in the present study. This
equation indicates that excretion of PD increased linearly in response to an incremental
supply of PB with an equimolar recovery of 0.85 mol/mol. This 0.85 recovery rate was
identical to that reported by Verbic et al. (1990) for European steers after adjustment for
digestibility of the purines (0.91) in microbial cells infused in the form of pruteen. The 0.85
value is also consistent with more recent values of 0.86 and 0.84, respectively, reported by
Vagnoni et al. (1997) and Orellana Boero et al. (1999) for European cows. Irreversible loss
via salivary ¯ow into the rumen (Chen et al., 1989), or as direct secretion of PD into the gut
(Altman and Dittmer, 1961 cited by Chen et al., 1990), were suggested as possible nonrenal losses. Verbic et al. (1990) cautioned that the recovery rate obtained using intragastrically nourished animals in their experiment might not be applicable to animals receiving
normal diets. The cattle in the present experiment, and those of Orellana Boero et al.
(1999), were fed normal diets and recovery rates similar to those of Verbic et al. (1990)
were recorded.
The endogenous excretion rate of 7.15 (S.E. 4.18) mmol per day calculated as the
intercept was numerically close to the 9.19 mmol per day estimated directly using the
fasting procedure conducted prior to the current study using the same cattle (Liang et al.,
1998). The latter value, when adjusted to metabolic weight (275 mmol/kg0.75 per day), was
15% higher than the value of 236 mmol/kg0.75 reported by Orellana Boero et al. (1999), but
lower than the value of 385 mmol/kg0.75 suggested by Verbic et al. (1990). The lower values
obtained in the two recent experiments, as compared to that in Verbic et al. (1990), could be
due to differences in the experimental procedures, particularly that the animals in the
former experiments were fed a normal diet while those in Verbic et al. (1990) were
intragastically nourished.
4.4. The concentration of PD in plasma
The concentration of plasma allantoin and uric acid of KK cattle recorded in the present
experiment were higher than values reported by Balcells et al. (1992) for steers (118.3,
12.6 mmol/l, respectively) and dairy cows (184.5, 34 mmol/l, respectively) reported by
Giesecke et al. (1994). McAllan (1980) reported that when purine was infused into
the small intestine of steers in the free or bound form, plasma allantoin levels increased by
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O. Pimpa et al. / Animal Feed Science and Technology 92 (2001) 203±214
15±35% over pre-infusion levels. In the present study, apparent increases in plasma
allantoin and total PD concentrations with increased rate of purine infusion were detected.
4.5. The GFR, TL and RB
Chen et al. (1997) suggested that the accuracy of PD recovery rate could be improved by
incorporating factors, such as GFR into the equation that relates urinary PD to exogenous
purine supply. Brody (1945) stated that, for animals of different sizes of the same species,
creatinine excretion is directly proportional to liveweight. Based on the above assumption,
GFR were calculated in the present study as the relationship between urinary creatinine
excretion rate and plasma creatinine concentration. Results show that GFR was not
affected by rate of exogenous PB supply (Table 4). However, the inverse relationship
between PB supply and PD reabsorption (RB) is interesting as Chen et al. (1990) reported a
similar observation for sheep, explaining that during purine-free nutrition, endogenous
purine loss is completely replaced by PD. However, when the animal is given an increasing
exogenous supply, biosynthesis is gradually replaced by utilization (salvage) of exogenous
purines, and becomes completely inhibited with an abundant supply of exogenous purines.
However, whether a similar explanation could account for such trend in zebu cattle requires
further investigation.
5. Conclusions
The relationship between urinary PD excretion and estimated duodenal PB ¯ow in KK
cattle was linear, with an estimated urinary excretion rate of 0.85, consistent with values
reported for European cattle by several groups. Although the estimated endogenous
excretion differed slightly from those in the literature, the present value is within the
reported range.
Acknowledgements
The present study was jointly sponsored by the Ministry of Science, Technology and
Environment, Malaysia and Joint FAO/IAEA Division, under project entitle ``Development, Standardization and Validation of Nuclear based Technologies for measuring
Microbial Protein Supply in Ruminant Livestock for Improving Productivity''. The
preparation of rumen and duodenal cannula for the experimental cattle by Dr. M.Y.
Loqman, and Dr. G.K. Mohd Azam Khan is appreciated.
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