Effects of energy and protein supplementation on
microbial-N synthesis and allantoin excretion
in sheep fed guinea grass
T. Jetana
a, N. Abdullah
b,*, R.A. Halim
c,
S. Jalaludin
d, Y.W. Ho
aaInstitute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia bDepartment of Biochemistry and Microbiology, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia
cDepartment of Agronomy and Horticulture, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia
dDepartment of Animal Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Received 16 June 1998; received in revised form 5 March 1999; accepted 21 February 2000
Abstract
An experiment was conducted to determine the effects of two types of protein, soybean meal (SBM) and ®sh meal (FM); and two types of energy supplements, corn ¯our (CF) and paper pulp (PP) on in vivo digestibility of organic matter (OM), rumen fermentation pattern and dilution rate, rumen microbial-N synthesis and ¯ow of organic matter and nitrogenous compounds through the duodenum in four Merino rams with an average weight of 54.44.5 kg. The relationships between duodenal purine ¯ow and urinary allantoin and duodenal protein/energy (MJ rumen VFA per day) ratio were also investigated. The experiment was conducted in a 44 Latin square design with a 22 factorial arrangement of dietary treatments. The animals, ®tted with both rumen and duodenal cannulae were housed in individual crates and fed chopped fresh guinea grass ad libitum twice daily, 100 g molasses and one of the four dietary supplements: (i) 170 g FM268 g PP (FMPP); (ii) 170 g FM268 g CF (FMCF); (iii) 200 g SBM200 g PP (SBMPP); and (iv) 200 g SBM200 g CF (SBMCF). Each supplement, at varying rates of rumen degradability, was formulated to provide similar amount of N and gross energy. The results showed that rumen pHs were similar, ranging from pH 5.8 to 6.0, for all animals fed the different dietary supplements. Rumen ammonia concentration was signi®cantly (p<0.05) higher in animals fed SBM (170.2± 190.7 mg N lÿ1) than in animals fed FM supplement (166.8±170.2 mg N lÿ1). Rumen VFA
84 (2000) 167±181
*Corresponding author. Tel.:60-3-9486101, ext. 3649; fax:60-3-9430913.
E-mail address: norhani@fsas.upm.edu.my (N. Abdullah)
concentrations were similar (94±103 mM) but molar proportions of acetate and propionate were respectively lower and higher in treatment SBMCF, leading to a signi®cantly decreased acetate:propionate ratio in this treatment (3.6) compared to SBMPP (4.3). There were no signi®cant differences between treatments in rumen ¯uid dilution rate and rumen volume. Duodenal OM ¯ow was similar in PP supplemented diets, but differed signi®cantly (p<0.05) between protein supplements in the CF diets. Organic matter digestibility in the rumen was signi®cantly (p<0.05) higher in the CFFM diet. Moreover, animals fed SBM supplement showed higher total tract OM digestibility than animals fed FM supplement. The ¯ow of nitrogenous compounds to the duodenum, i.e., total-N, non-ammonia-N and rumen-undegradable-N were not signi®cantly affected by either protein or energy supplements. Microbial-N ¯ow tended (p<0.08) to be higher in sheep fed CF supplement (average of 10.2 g per day) than in those fed PP supplement (average of 8.1 g per day). Urinary allantoin excretion was low (0.30±0.42 mmol per day/kg BW0.75). A positive linear
correlation (r0.73,p<0.005) between urinary allantoin (mmol per day/kg BW0.75) and duodenal purines (mmol per day/kg BW0.75) was observed. The ef®ciency of rumen microbial-N synthesis
based on OM truly digested in the rumen was signi®cantly (p<0.02) higher in sheep fed CF supplement (15.2±16.6 g N kgÿ1OMTDR) than in those fed PP supplement (12.2±12.8 g N kgÿ1
OMTDR). Differences in microbial protein:energy ratio or total duodenal protein:energy ratio among dietary treatments were not signi®cant.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Sheep supplementation; Digesta ¯ow; Allantoin excretion; Microbial protein; Energy; Protein; Guinea grass
1. Introduction
It is generally accepted that tropical forages are of low quality and intake of forages by animals is usually limited by the high level of ®bre and low nutrient content, particularly proteins which are essential for both animal and rumen microbial growth. Supplementary feeding as a means of increasing the nutritive value of these forages has been practised to promote animal production. Protein supplements can increase forage intake and digestibility and improve performance of animals (Minson, 1990; Poppi and McLennan, 1995). However, protein supplement may be ineffectively utilized in the rumen if appropriate energy sources are not available. Under such a condition, protein breaks down into amino acids and undergo deamination process. The ammonia-N generated is absorbed and excreted in the urine in the form of urea (Nolan, 1993).
In energy-de®cit diets, additional energy input is necessary to optimise protein synthesis (Poppi and McLennan, 1995). The slow degradation of ®bre by cellulolytic bacteria in the rumen requires a good synchrony between energy produced and ammonia-N release (Beever and Siddons, 1986). Ways to increase cellulolytic bacterial activity will improve digestion and increase protein microbial supply to the host animal. Supplementing the rumen with protein and energy sources will enhance microbial growth and digestion, and thus increase protein and energy supplies to the animal.
The objective of this study was to determine the effects of protein and energy supplements that have different rates of degradation in the rumen on microbial activity and synthesis. The experiments carried out were in vivo digestibility, estimation of digesta ¯ow and rumen microbial protein synthesis, rumen ¯uid characteristics, rumen
¯uid dilution rate and rumen volume. The relationship between duodenal purine ¯ow and allantoin excretion in urine and the ef®ciency of microbial protein synthesis were also investigated.
2. Materials and methods
2.1. Animals, diets and experimental designs
Four Merino rams with an average weight of 54.44.5 kg were each ®tted with a rumen cannula and a re-entrant duodenal cannula at ca. 4 cm posterior to the pylorus. The surgical procedure was the same as that described by Ivan and Johnston (1981). The sheep were dosed to kill gastrointestinal parasites 4 weeks before surgery. After the operation, the rams were housed in individual pens and intensively cared for 5 weeks before used.
The study was conducted using 44 Latin square design with a 22 factorial arrangement of dietary treatments. The animals were fed twice daily with chopped guinea grass (Panicum maximum) (5 cm) ad libitum and the protein (®sh meal or soybean meal) and energy (paper pulp or corn ¯our) supplements. The supplements offered on fresh weight basis per day per animal are presented in Table 1. Each supplement was formulated to provide similar amount of N and gross energy but with varying rates of rumen degradability. Corn ¯our was purchased from Glow San Sdn Bhd, Selangor,
Table 1
Liveweight of sheep and feed supplements (g fresh weight) offered daily Itema Dietary treatments
Fish meal Soybean meal
Paper pulp Corn flour Paper pulp Corn flour Liveweight (kg) 53.5 55.0 54.8 54.1 Feed supplement:
®sh meal 170 170 0 0
soybean meal 0 0 200 200
paper pulp 268 0 200 0
corn ¯our 0 268 0 200
molasses 100 100 100 100
Daily amounts of different constituents of the supplements offered per animal:
Dry matter (g) 470 460 439 431
Organic matter (g) 406 396 421 413
Nitrogen (g) 13.0 12.7 13.1 12.8
Starchb(g) 0 228 0 170
Gross energyc(MJ) 7.4 7.2 7.4 7.3
aGuinea grass was fed ad libitum (guinea grass contained 91% DM, 93% OM and 1.7% N). bStarch was analysed by the method described by Southgate (1976).
cGross energy was determined by using IKA-Bomb Calorimeter System C 4000 A adiabatic (Germany).
Malaysia. Paper pulp boards made from soft wood (Scott Paper Sdn. Bhd.) were cut into small pieces (0.50.5 cm) by using an electric plane.Fish meal and soybean meal were purchased from Mansura Trading, Selangor, Malaysia.
The experiments were carried out in four periods. Each period lasted for 26 days, where the ®rst 14 days were for dietary adaptation and the last 12 days for experimentation. The experiments carried out were in vivo digestibility, estimation of digesta ¯ow and rumen microbial protein synthesis, rumen ¯uid characteristics, rumen ¯uid dilution rate and rumen volume.
2.2. In vivo digestibility
The animals were placed (2 days before the collection period) in individual crates ®tted with containers for urine and feces collection. The amounts of grass refusal (no supplement refusal for each animal throughout the four experimental periods), feces and urine outputs were recorded twice daily for 9 days. Ten percent representative aliquots of the supplements and fresh guinea grass offered and 15% aliquots of guinea grass refusals and fecal outputs were collected and stored atÿ208C. Total urine outputs were acidi®ed (pH 2±3) by collecting into containers containing50 ml of 50% HCl and a subsample of urine (100 ml) was taken and kept at ÿ208C. Daily individual acidi®ed urine samples were analysed for total-N using the micro Kjeldahl method and for allantoin using the Rimini±Schriver color reaction method (Pentz, 1969).
Frozen samples of supplements and guinea grass offered, guinea grass refused and fecal outputs were thawed, weighed and dried at 608C in an oven for 72 h. Individual dried samples were ground through a 2 mm screen, and kept in containers for ash, organic matter (OM) and protein analyses.
2.3. Digesta ¯ow and rumen microbial protein synthesis
Digesta ¯ow (passing through the duodenum) was determined by the double marker technique of (Faichney, 1980, 1993) and microbial-N estimated from the purine content of duodenal digesta and rumen microbial fractions (Zinn and Owens, 1986). Chromium mordanted paper pulp was used as the particulate marker, while cobalt EDTA was used as the liquid phase marker. Both markers were prepared according to the method described by UdeÂn et al. (1980). Ten grams of chromium mordanted paper pulp (140 mg Cr per day) were given to each animal from day 12 to 23 by mixing with the morning supplements, while cobalt EDTA (180 mg Co per day) was continuously infused through the rumen cannula at 5 ml hÿ1from day 14 to 23 with a multi-channel peristaltic pump (Miniplus, Gilson, France).
Duodenal digesta samples were obtained at 12.00 a.m., 06.00 a.m., 12.00 p.m. and 06.00 p.m. (Day 22) and at 03.00 a.m., 09.00 a.m., 03.00 p.m. and 09.00 p.m. (Day 23). One hundred millilitre of duodenal digesta were collected from a re-entrant duodenal cannula by letting the digesta ¯ow out by gravity through a plastic tube into a container. Duodenal samples were stored atÿ208C for analysis.
The eight duodenal samples from each animal at each period were thawed and pooled by taking equal portion from each sample. A total of 16 (4 animals, 4 periods) pooled
samples were obtained. Each pooled sample was homogenized and then divided equally into two portions. One portion was used as a representative of pooled whole duodenal digesta phase, the other portion was centrifuged at 1000g for 5 min, and the pellet obtained represented the pooled particulate duodenal digesta phase. Both phase samples were lyophilized and analysed for ash, OM and total N by standard procedures (AOAC, 1984). To determine non-ammonia-nitrogen (NAN), the samples were made alkaline with saturated tetraborate solution (pH 9), then boiled (for 3 min only) to remove ammonia and the remaining duodenal digesta was subjected to crude protein (N) analysis by Kjeldahl digestion and titration. Purine bases were determined by the method described by Zinn and Owens (1986) using yeast RNA as the standard. Chromium and Co concentrations were measured by the method described by Le Du and Penning (1982).
2.4. Rumen ¯uid characteristics
Approximately 150 ml of rumen ¯uid were taken from the middle part of the rumen by using a 60 ml hand syringe at the same sampling times as the duodenal digesta collections. The pH values of the rumen ¯uids were immediately recorded with an electrode pH meter. Rumen ¯uid samples were divided into two portions. One portion was used for microbial isolation, where 20 ml of 13.7% formaldehyde in normal saline (0.9% NaCl) were added to 100 ml rumen ¯uid and stored frozen. Another portion was used for ammonia-N and VFA analyses where 10 ml of 24% metaphosphoric acid in 12 M sulphuric acid were added to 50 ml of rumen ¯uid. The mixture was stored at ÿ208C prior to ammonia-N and VFA analyses (Jetana et al., 1998).
Bacteria were isolated from the rumen ¯uid for estimation of bacterial dry matter, ash, nitrogen and purines bases. To isolate bacteria from rumen ¯uid, the eight rumen ¯uid samples (each animal and each period) were thawed and pooled in a similar manner to the duodenal samples described above. Pooled rumen ¯uids were centrifuged at 600gfor 5 min. The supernatant was decanted into a new tube and re-centrifuged as above. The supernatant was again transferred into a new tube and centrifuged at 28,000g for 20 min. The bacterial pellet obtained was washed twice with saline (0.9% NaCl) and centrifuged at 28,000g for 20 min. The pellet was lyophilized and analysed for OM, crude protein and purine bases using the same methods described above.
2.5. Rumen ¯uid volume and dilution rate
After Co-EDTA infusion was terminated on Day 24, rumen contents were collected immediately (0 h) and then at 2, 8, 16, 20, 32 and 50 h. The samples were kept atÿ208C for Co analysis to calculate the rumen ¯uid dilution rate and rumen volume. Cobalt concentration was determined using the method described by Okine et al. (1989).
2.6. Calculation
The true nutrient ¯ow through the duodenum was calculated by the double marker method of (Faichney, 1980, 1993) where cobalt EDTA and chromium mordanted paper pulp were used as the liquid phase marker and the particulate phase marker, respectively.
The equations developed by Czerkawski (1986) were employed to calculate the total tract digestibility and true digestibility of OM in the rumen. The rumen liquid dilution rate was determined as the slope of the regression of the natural logarithm of the marker concentration against time, ®tted to a ®rst order model.
2.7. Statistical analysis
The means of each parameter measured in the digestibility studies, nutrient intake and ¯ow were analysed by the analysis of variance (ANOVA) techniques using the general linear model (GLM) procedures of the Statistical Analysis System Institute (Statistical Analysis Systems Institute, 1988). Treatment means were compared by the least signi®cant difference method (LSD).
The 22 factorial analysis of variance was used to examine the effects of protein and energy sources and their interactions.
Rumen ¯uid data (pH, ammonia-N and VFA) were analysed by split-plot analysis of variance (Snedecor and Cochran, 1967) using the following model:
YijklmmAiPjTkeijkHi AHil PHjl THkleijklm
where m is mean of A, P, T and H for animal, period, treatment and time effects, respectively;eijkthe main plot error;eijklmthe sub-plot error.
The main plot included dietary treatment, period and animal effects: treat-mentperiodanimal was used in the Statistical Analysis Systems Institute, (1988) model to calculate main-plot error, and the subplot was tested for time and timetreatment interaction. Timetreatment interaction when detected (p<0.05) is shown in the results.
3. Results
The chemical compositions of guinea grass and corn ¯our (CF), paper pulp (PP), soybean meal (SBM) and ®sh meal (FM), and the amounts of supplements provided daily per animal have been described earlier by Jetana et al. (1998). Guinea grass contained 1.7% N (10.4% crude protein) and 17.3 kJ gÿ1gross energy. Corn ¯our contained 85% starch, while PP contained 92.9% NDF. Both energy supplements (i.e., CF and PP) had trace amounts of N (<0.1%). Soybean meal contained 6.7% N and ®sh meal contained 8.0% N.
The animals were fed protein supplements at 12.7±13.1 g N per day. However, the amount of starch provided by the supplements ranged from 0 in PP to 228 g in CF. Similarly, NDF content varied widely between the diets. It was high in PP supplemented diet (232±237 g) but much lower in CF supplemented diet (1.3±65.1 g).
Table 2 shows the effects of protein and energy supplements on rumen ¯uid characteristics. Rumen pH did not differ signi®cantly between treatments. However, rumen ammonia-N was affected (p<0.05) by protein sources. Sheep fed SBM supplement had higher (p<0.05) rumen ammonia-N concentrations than those fed FM, but energy sources had no signi®cant effect. Total VFA concentration (ranging from 94.1 to
102.5 mM) was not signi®cantly affected by dietary treatments. The effects of both energy and protein sources were apparent in both acetate and propionate molar proportions. Molar proportion of acetate was signi®cantly (p<0.05) higher in sheep fed FMPP (73.3%) than in sheep fed SBMCF (70.1%). However, molar proportion of propionate was affected (p<0.05) by energy source only in sheep fed SBM supplement, being higher when supplemented with CF (19.9%) than with PP (17.4%). As a result, the acetate to propionate ratio differed between energy supplements only in the SBM diets, where sheep fed CF had a signi®cantly lower ratio (3.6) than those fed PP (4.3).
Molar proportions of butyrate tended (p<0.06) to be higher in sheep fed SBM diets (average of 8.1%) than in sheep fed FM diets (average of 7.8%). The molar proportions of isobutyrate, isovalerate, valerate and caproate made up <2% of the total acids.
Rumen ¯uid dilution rates and rumen volumes determined by using Co-EDTA as a marker were not signi®cantly affected by dietary treatments.
Table 3 shows the chemical compositions (% of DM) of bacteria isolated from rumen ¯uid. The average amount of total purine bases (RNA equivalent) was 5.30.4% and the ratio obtained for RNA equivalent-N to total bacterial-N was 11.6%.
The effects of diets on organic matter intake (OMI), OM ¯ow rates at the duodenum and OM digestibility are shown in Table 4. The amounts of OM (from guinea grass and supplements) consumed by the animals in the different dietary treatments were similar, averaging 95691 g per day. Duodenal OM ¯ow was similar in PP supplemented diets but differed signi®cantly (p<0.05) between protein supplements in the CF diets. The total OM ¯ow through the duodenum decreased when the CF diet was supplemented with FM compared to SBM and the same trend was also noticed in the true OM ¯ow because microbial OM did not vary between protein supplements. As a result, the proportion of
Table 2
Rumen pH, rumen ammonia-N and volatile fatty acid (VFA) concentration, rumen ¯uid dilution rates and rumen volumes of sheep fed guinea grass as a basal diet with different protein and energy supplements
Parameter Fish meala Soybean meala S.E.D.b Probability for contrast between acetate:propionate ratio 4.1 4.2 4.3 3.6 0.2 0.04 0.001d
Rumen ¯uid dilution rate (l hÿ1) 0.07 0.08 0.08 0.07 0.01 NS NS Rumen volume (l) 4.7 4.5 4.8 6.0 1.0 NS NS
aMeans in the same row with different letters are signi®cantly different (p<0.05). bStandard error of difference.
cNot signi®cantly different (p>0.05).
dSigni®cant interaction between protein and energy (p<0.05).
OM digested in the rumen was higher (p<0.05) in the CFFM diet than in the rest of the treatments. Total tract OM digestibility was not signi®cantly different between energy supplements, but a signi®cant (p<0.05) overall effect of protein sources was observed, where sheep fed SBM supplement had higher digestibility values than sheep fed FM supplement. Fecal OM output were not signi®cantly different among the dietary treatments.
The intake of N by sheep and the ¯ow of nitrogenous compounds from the rumen are shown in Table 5. Nitrogen intakes were similar (in the range of 22.5±24.3 g per day) for all animals fed the different diets. The ¯ow of nitrogenous compounds through the
Table 3
Chemical composition of pooled mixed rumen bacteria (% DM) and ratios between RNA equivalent-N and mixed rumen bacteria-N of sheep fed guinea grass as a basal diet with different protein and energy supplements Parameter Fish meal Soybean meal Means SD
Paper pulp Corn flour Paper pulp Corn flour
Organic matter 83.6 79.2 84.2 83.6 82.6 2.0
Nitrogen 7.8 7.4 7.0 6.6 7.3 0.4
Ash 15.5 20.1 15.1 15.7 16.6 2.0
Purine bases (RNA equivalent)a 5.7 4.6 5.5 5.3 5.3 0.4
RNA equivalent-N:total-N in mixed rumen bacteria (%)
11.5 9.8 12.4 12.6 11.6 1.1
aYeast-RNA was used as the standard to calculate total purines and yeast RNA-N was 15.7% (n4).
Table 4
Organic matter intake (OMI), digestibility and ¯ow of OM from the rumen of sheep fed guinea grass as a basal diet with different protein and energy supplements
Parameter Fish meala Soybean meala S.E.D.b Probability for
contrast between guinea grass 511 570 535 572 129 NSc NS
total 917 966 956 985 129 NS NS
Duodenal ¯ow (g per day):
total OM 405 ab 350 b 397 ab 495 a 47.5 0.09 NSd microbial OM 91.5 113.8 93.5 130.0 17.5 NS 0.06 non-microbial OM 314 ab 237 b 304 ab 365 a 44.09 NS NS Faecal OM output (g per day) 343 335 318 326 34.4 NS NS OM truly digested in the rumen (%) 65.0 b 74.6 a 68.2 b 64.0 b 2.5 0.08 NSd Total tract OM digestibility (%) 65.3 65.6 66.9 67.7 2.2 0.05 NS
aMeans in the same row with different letters are signi®cantly different (p<0.05). bStandard error of difference.
cNot signi®cantly different (p>0.05).
dSigni®cant interaction between protein and energy (p<0.05).
duodenum, i.e., total-N, non-ammonia-N (NAN) and rumen-undegradable-N were not signi®cantly (p>0.05) affected by either protein or energy supplements.
A slightly higher amount of total purine bases (RNA equivalents) passed through the duodenum in animals fed CF supplement (average of 7.1 g per day) than in those fed PP (average of 6.2 g per day), although differences did not reach statistical signi®cance. Moreover, they were re¯ected in the estimated microbial-N ¯ow through the duodenum which tended to be higher (p<0.08) in sheep fed CF supplement (average of 10.2 g per day) than in those fed PP supplement (average of 8.1 g per day). There was no signi®cant effect of protein supplements on the ¯ow of purine bases or microbial-N through the duodenum.
Non-ammonia±non-microbial-N (NANM-N) ¯ow was similar in PP supplemented diets but differed signi®cantly (p<0.05) between protein supplements in the CF diets.
Table 5
Nitrogen (N) intake and ¯ow of nitrogenous compounds from the rumen of sheep fed guinea grass as a basal diet with different protein and energy supplements
Parameter Fish meala Soybean meala S.E.D.b Probability for contrast between
total-N 18.6 18.7 18.0 19.4 1.6 NS NS non-ammonia-N (NAN) 16.3 15.2 15.3 17.7 1.1 NS NS bypass-Nd 4.0 1.2 3.8 4.3 1.2 NS NS
purine bases (RNA equivalent) 6.2 6.6 6.2 7.7 1.1 NS NS purine bases (mmol per day)e 6.8 7.3 6.8 8.5 1.2 NS NS
microbial-Nf 8.5 10.8 7.8 9.7 1.4 NS 0.08
non-ammonia±non-microbial-N (NANM-N)
7.7 a 4.5 b 7.5 a 8.0 a 1.1 0.07 0.1 NAN/N intake ratio 0.75 0.66 0.65 0.73 0.1 NS NS Rumen-N degradability (%) 81.8 94.3 84.3 83.8 4.12 NS NS Faecal-N out put (g per day) 9.4 9.7 9.0 8.0 1.2 NS NS
aMeans in the same row with different letters are signi®cantly different (p<0.05). bStandard error of difference.
cNot signi®cantly different (p>0.05).
dBypass-NNAN±microbial-N±endogenous-N (where endogenous-N was estimated to be 181 mg/kg BW0.75
(érskov et al., 1986).
eCalculated based on 1.107 mmol purine/g yeast RNA (Balcells, personal communication). fCalculated based on the method described by Zinn and Owens (1986).
Sheep fed FMCF had signi®cantly (p<0.05) less (4.5 g per day) NANM-N ¯ow than sheep fed other diets (7.5±8.0 g per day).
The ratio of NAN passing through the duodenum to total-N intake and the amount of NAN expressed in terms of crude protein (CP) per kg of digestible organic matter intake (DOMI) were not signi®cantly different among sheep fed the different supplements. Rumen-N degradability or the amount of N that is degraded in the rumen compared to the amount of N consumed (calculated by using the equations described by ARC, 1980; Sinclair et al., 1993) was similar for sheep given the different diets. The values were within the range of 81.8±94.3%.
Fecal-N output, urine-N excretion, N-balance of sheep and allantoin excretion in urine were not signi®cantly (p>0.05) different among sheep given the different dietary treatments. The sheep were in positive N-balance. Urinary allantoin was in the range of 0.30±0.42 mmol per day/kg BW0.75for the four dietary treatments.
The results on ef®ciency of microbial-N synthesis in the rumen, the ratios of microbial protein (MP) and the total duodenal protein (DP) to the energy (E) measured in terms of MJ per day VFA in the rumen are presented in Table 6.
The ef®ciency of rumen microbial-N synthesis (MNeff) based on organic matter truly digested in the rumen (OMTDR) was signi®cantly (p<0.02) different among energy supplements. Sheep fed CF had higher MNeff compared to sheep fed PP diets.
The energy values of VFA (MJ) produced in the rumen were calculated by multiplying the total VFA with OM digested in the rumen (Czerkawski, 1986). The energy values calculated were almost similar for all animals fed the different diets. They were in the range of 4.6±5.9 MJ per day. Differences observed in the microbial protein:energy (MP/ E) ratios or the total duodenal protein:energy (DP/E) ratios among dietary treatments were not signi®cant, although the ratios were higher in sheep fed FMPP and SBMCF supplements.
Table 6
Microbial-N synthesis based on organic matter truly digested in the rumen and protein:energy ratio of sheep fed guinea grass as a basal diet with different protein and energy supplements
Parameter Fish meala Soybean meala S.E.D.b Probability for contrast between Microbial-N (g kgÿ1OM truly
digested in the rumen)
12.8 b 15.2 ab 12.2 b 16.6 a 1.42 NSc 0.02
VFA produced (MJ per day) (E)d 4.6 5.8 5.9 4.8 0.7 NS NS
MP/Ee 13.0 12.5 8.5 13.2 2.8 NS NS
DP/Ef 24.5 18.7 16.8 24.0 4.0 NS NS aMeans in the same row with different letters are signi®cantly different (p<0.05).
bStandard error of difference. cNot signi®cantly different (p>0.05).
dCalculation based on OM truly digested in the rumen (Czerkawski, 1986). eMicrobial protein6.25microbial-N from Table 5.
fTotal duodenal protein6.25(endogenous-Nmicrobial-Nbypass-N) from Table 5.
4. Discussion
Sheep fed the four dietary treatments in the present study had similar total OM and N intakes as the supplement allowance was ®xed to supply similar amounts of N and OM and grass intake by the animals did not vary between treatments.
4.1. Rumen parameters
The means of rumen pH for all the dietary treatments ranged from 5.8 to 6.0. These values are below pH 6.2, where optimum cellulolytic activity takes place (Stewart, 1977). However, a rumen fermentation with a high acetate production was observed for all diets in this study, although the average ratio of concentrate to roughage in terms of OM was 44:56. This pattern of high acetate production is typical of a high forage diet that encourages the growth of cellulolytic bacteria, which produce mainly acetate. A signi®cant (p<0.05) interaction between protein and energy sources was observed in acetate proportion, where sheep fed FMPP supplements had higher proportions than sheep fed SBMCF supplements. This is congruent with the nature and degradability rates of the protein and carbohydrate supplements used. Propionate proportion was signi®cantly (p<0.05) affected by energy source only in the SBM fed sheep and was related to the presence of a readily fermentable carbohydrate (starch) in CF.
The means of rumen ammonia-N were higher than 100 mg N lÿ1 in sheep fed both protein sources. These concentrations are within the range of 100±200 mg N lÿ1required for optimum digestion (Krebs and Leng, 1984; Leng, 1990). Sheep supplemented with SBM showed higher rumen ammonia-N concentration than sheep supplemented with FM. Oldham et al. (1977) have suggested that sheep fed FM supplement generally have low concentrations of rumen ammonia-N concentration because FM is a low rumen degradable protein. However, percentage values of rumen degradable nitrogen (Table 5) were not signi®cantly different among all dietary treatments, with the value for FM supplemented sheep higher than expected. This is probably due to the improved digestion and utilisation of dietary protein in the presence of easily degradable carbohydrate supplements as indicated by the increased in OM digestion in the rumen of sheep fed FM supplement (Table 4).
4.2. Duodenal ¯ows
Microbial-N ¯ow was not affected by protein supplements, but tended (p<0.08) to increase in CF diets. This may re¯ect the effect of energy substrates on bacterial growth rate on N non-limiting conditions.
The ef®ciency of rumen microbial-N synthesis was signi®cantly higher in sheep fed CF supplement, where the values ranged from 15.2 to 16.6 g N kgÿ1OMTDR. These values were lower than 19.3 g N kgÿ1OMTDR estimated by Czerkawski (1986) from a number of published data. However, ef®ciency of microbial-N synthesis in the rumen had been observed to vary between 10 and 70 g N kgÿ1OMDR (Van Nevel and Demeyer, 1977). Variability in ef®ciency of microbial-N synthesis exists as a result of various factors like concentration and sources of nitrogen and carbohydrates, rumen dilution rate and
frequency of feeding (Stern and Hoover, 1979) and the type of microbial markers used (Perez et al., 1996a).
The amount of non-ammonia±non-microbial-N (NANM-N) which indicates the amount of undegraded and endogenous-N transferred to the intestine was lower in sheep fed FMCF diet, which is in agreement with the high N degradability and microbial-N synthesis recorded on this treatment. This is rather surprising as FM is expected to be less degradable and utilised in the rumen. It appears that both supplements are better synchronised in terms of energy and N released for microbial protein synthesis.
The ratio of NAN passing through the duodenum to total-N intake or N transfer to the intestine in sheep was found to be in the range of 65±75%, i.e., lower than N intake. This differs from the results reported by Egan et al. (1975) and Schloesser et al. (1993) in which NAN transferred to the intestine was higher than N intake. The latter result which often occurs in low quality diets, is probably a consequence of recycling of rumen ammonia-N. In the present study, the guinea grass used as the basal diet was a medium quality protein source (1.7% N) which probably supplied suf®cient N to meet rumen requirements as indicated by the high rumen ammonia-N concentration in the rumen of sheep fed the four different diets (167±191 mg N lÿ1, Table 2).
4.3. Allantoin excretion
Excretion of allantoin in the urine was low in sheep fed the different diets. There was no signi®cant (r0.5) linear correlation between the amount of allantoin excreted in urine and microbial-N synthesis in the rumen, but a signi®cant linear correlation (r0.73;
p<0.005; Fig. 1) between total purine at the duodenum and excretion of allantoin was observed. The equation obtained from this relation is:
Urinary allanotin mmol per day 0:863X30:37
where X is total purine ¯ow through the duodenum (mmol per day/kg BW0.75). The recovery coef®cient was similar to the value reported by Balcells et al. (1991) who also observed a relationship between allantoin excretion in urine and microbial purine infused at the duodenum of sheep. However, in this study, only allantoin was used to derive the relationship. In sheep, substantial amount of other purine derivatives are also excreted. Chen and Gomes (1995) reported the proportions to be 60±80, 30±10 and 10±5% for allantoin, uric acid, xanthine and hypoxanthine, respectively. Hence, the recovery coef®cient would be enhanced if total purine derivatives was used.
The reason for the lack of a signi®cant linear correlation between allantoin excretion in the urine and microbial-N synthesis in the rumen could be due to the variability in the conversion factor (RNA equivalent-N:bacterial-N) used in calculating the microbial-N. Perez et al. (1998) reported signi®cant differences in purine bases:N ratio between liquid-associated bacteria (LAB) and solid-liquid-associated bacteria (SAB) in the rumen. Fractional contribution of LAB and SAB to the postruminal bacteria was signi®cantly in¯uenced by diets (Perez et al., 1998) and this would affect the microbial-N estimates when either LAB or SAB was used as the reference. Microbial-N might be overestimated when purine bases were used as the only marker. Perez et al. (1996b) had reported that the total purine
bases estimated from duodenal contents may not be totally of microbial origin as non-microbial nucleic acids may contribute 11±23% for SBM and 20±40% for FM.
The results demonstrated a positive linear relationship between allantoin excretion and microbial purine ¯ow at the duodenum. Microbial-N ¯ow and ef®ciency of microbial-N synthesis based on OMTDR was better in animals fed CF compared to PP supplements.
Acknowledgements
The funds provided by the ministry of Science, Technology and the Environment of Malaysia under the Intensi®cation of Research Priority Areas (IRPA) Programme (Project Code 1-07-05-038) are acknowledged.
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