Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol83.Issue3-4.Mar2000:
Animal Feed Science and Technology
83 (2000) 261±272
Colonization and source of N substrates used by
microorganisms digesting forages incubated in
synthetic ®bre bags in the rumen
R.M. Dixon*,1, S. Chanchai2
School of Agriculture and Forestry, The University of Melbourne, Parkville, Vic 3052, Australia
Received 13 April 1999; received in revised form 5 October 1999; accepted 11 November 1999
Abstract
Six mature sheep were fed restricted amounts of either a medium quality roughage or a 1 : 1
mixture of the roughage and barley grain. Disappearance of DM of three roughages (barley straw,
oat hay and lucerne hay) from synthetic ®bre bags incubated in the rumen for 6 and 24 h was
determined. Also, during intraruminal infusions of 15NH4C1, synthetic ®bre bags containing each of
the three roughages were incubated in the rumen for 6 and 24 h. The origins and amounts of
adherent microbial N associated with the bag residues after incubation and washing were estimated
from the 15N enrichments of rumen ammonia, adherent microbial N and bag residue total N. The
proportion of adherent microbial N derived from the rumen ammonia pool was not affected
(p > 0.05) by diet, but was lower (p < 0.05 or p < 0.01) for microorganisms adherent to lucerne hay
bag residues (26 and 33% at 6 and 24 h, respectively) than microorganisms adherent to barley straw
(47 and 77% at 6 and 24 h, respectively) or oat hay bag residues (44 and 80% at 6 and 24 h,
respectively). The proportion of bag residue N consisting of microbial N was not affected (p > 0.05)
by the diet, but was lower (p < 0.01) in lucerne hay bag residues (54 and 69% at 6 and 24 h,
respectively) than in barley straw or oat hay bag residues (75±76% at 6 h and 81% at 24 h).
Microbial N remaining associated with bag residues ranged from 3.7 to 7.6 mg microbial N/g
residual DM. Because of this microbial N associated with bag residues, rumen degradability of
lucerne hay N was underestimated by ca. 12 and 4% at 6 and 24 h, respectively. The
underestimation of the rumen degradability of oat hay N was more than 26% units, and that of
barley straw N was more than 75% units. In conclusion, this experiment indicated that the
microorganisms digesting low N forages are much more dependent on rumen ammonia as a N
*
Corresponding author. Tel.: 61-747-849170; fax: 61-747-849232.
E-mail address: dixonr@prose.dpi.qld.gov.au (R.M. Dixon).
1
Present address: Queensland Beef Industry Institute, Swan's Lagoon Research Station, Millaroo, Ayr, Qld
4807.
2
Present address: Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand.
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 1 2 8 - 5
262
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
substrate than those digesting high N forages, and that microbial N associated with the residues
remaining in synthetic ®bre bag residues following incubation and washing was substantial.
# 2000 Elsevier Science B.V. All rights reserved.
Keywords: Sheep; Synthetic ®bre bags; Microbial colonization
1. Introduction
The nitrogen (N) requirements of ruminants are usually considered in terms of the
nitrogenous substrates for rumen microorganisms (rumen degradable protein (RDP) or
effective rumen degradable protein (ERDP)), and dietary protein escaping rumen
fermentation but digested in the small intestine (SCA, 1990; AFRC, 1993). Since most
ERDP is utilised as a microbial substrate after its conversion to ammonia (Leng and
Nolan, 1984; Morrison and Mackie, 1996), an understanding of the requirements of
microorganisms for ammonia N to ferment various classes of feedstuffs is essential for
understanding dietary requirements for ERDP. Current feeding standards assume that the
amount of ERDP required is, with some corrections, equivalent to the amount of net
microbial protein synthesised and that the latter is a function of the availability of
fermentable energy in the rumen. However, the concentrations of rumen ammonia
required for the maximum rate of fermentation and voluntary intake of ®brous feeds, and
the adverse effects of low concentrations, appear to be greater for low quality forages
(Alvarez et al., 1984; Krebs and Leng, 1984; Perdok et al., 1988). In addition, the extent
to which microorganisms digesting forages depend on rumen ammonia as a N substrate
appears to vary with the protein content of the forage (Nolan et al., 1976; Neutze et al.,
1986; Dixon, 1999).
Disappearance of the nitrogenous components of feedstuffs from synthetic ®bre bags
incubated in the rumen has been accepted as a standard procedure to evaluate the N
degradability and ERDP content of feedstuffs (SCA, 1990; AFRC, 1993). However, any
microbial N remaining in synthetic ®bre bag residues after washing results in
underestimation of the degradability of the dietary protein. The underestimation is small
for protein meals (Mathers and Aitchison, 1981; Varvikko and Lindberg, 1985; Gonzalez
et al., 1998) but a number of experiments indicate that it is substantial for forages.
Microbial N remaining in residues from forages incubated in synthetic ®bre bags has
been measured using 35S tracers (Mathers and Aitchison, 1981; Kennedy et al., 1984),
diaminopimelic acid (Nocek and Grant, 1987; Olubobokun et al., 1990; Alexandrov,
1998), D-amino acids (Rooke et al., 1984), or by dilution of 15N tracer incorporated into
the plant material (Varvikko and Lindberg, 1985; Wanderley et al., 1993). However, there
may be substantial inaccuracies in these estimates due to dif®culties with methods used to
measure the microbial N in synthetic ®bre bag residues.
The present experiment investigated the extent to which adherent microorganisms
digesting forages ranging in N content utilized the rumen ammonia pool as a N substrate.
A second objective was to measure microbial N remaining in residues of forages
incubated in synthetic ®bre bags using an 15N tracer procedure which avoided some of the
dif®culties associated with the procedures used in previous experiments. Measurements
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
263
were made for three hays ranging in N content and in sheep fed a forage or a mixed
forage±cereal grain diet.
2. Materials and methods
2.1. Sheep,
15
NH4Cl infusions and synthetic ®bre bags
Six mature Merino sheep (liveweight mean 48, range 43±53 kg; 2±3 years of age)
surgically prepared with rumen cannulas were held indoors in metabolism crates. Three
sheep were allocated at random to each of two diets consisting, on an air dry basis, of
500 g chopped oat hay and 500 g chopped lucerne hay (Diet 1), or 250 g chopped oat hay,
250 g chopped lucerne hay and 500 g whole barley grain (Diet 2). From day 7 the diets
were fed from continuously moving belts and the room was continuously lit.
On days 16 and 18 duplicate synthetic ®bre bags containing barley straw, oat hay or
lucerne hay were inserted into the rumen at 0900 h and removed after 6 and 24 h. The
bags (50 120 mm, 44 mm pore size mono®lament nylon cloth (Swiss Screens,
Moorabbin, Victoria, Australia)) contained ca. 2.0, 2.3 and 2.5 g of barley straw, oat
hay and lucerne hay, respectively, and also a 3.5 g metal ball as a weight. These hays were
ground through a 1 mm screen (Makla mill, Crompton Parkinson model 8302A-P,
Australia). On removal from the rumen bags were brie¯y washed and then stored at 58C
until all bags had been removed. The bags were then washed manually under running
water until the washings were clear and dried at 708C. The solubility of dry matter (DM)
of the hays was also determined by soaking ®ve bags containing each ground hay in
0.15 M NaCl for 3 h before washing and drying.
Intra-ruminal infusions of 15NH4C1 (30 mg 15N in 850 ml aqueous solution per day,
98.9 atom% excess, Amersham International, UK) were commenced at 0900 h on day 22
and were continued for 96 h. Samples of rumen ¯uid (50 ml) were obtained by suction
through a gauze covered cage suspended in the ventral sac of the rumen before infusion of
15
N tracer commenced and also six times from 48 to 96 h of the infusions. The samples
were acidi®ed (0.5 ml 5 M H2SO4) and were then centrifuged (1000 g for 1 min). The
supernatant thus obtained was again centrifuged (14 000 g for 15 min) to separate
rumen ¯uid free of particulate matter and a microbe-rich fraction (Nolan, 1972). The
microbe-rich fraction was washed once with 0.15 M NaCl, isolated again by
centrifugation (14 000 g for 15 min) and then stored frozen pending 15N analysis.
On days 24 and 25, during the intra-ruminal 15NH4Cl infusions, synthetic ®bre bags
containing the barley straw, oat hay and lucerne hay were inserted at 0900 h and were
removed after 6 and 24 h. The procedures for these synthetic ®bre bags were as described
above, except that the bags contained 4.0, 4.6 and 5.0 g barley straw, oat hay and lucerne
hay, respectively, and the bags (85 160 mm) were made from a 100 mm pore size
mono®lament nylon cloth (Swiss Screens, Moorabbin, Victoria, Australia). Following
washing the bags were stored at 58C. Microorganisms adherent to the residues remaining
in the bags following incubation and washing were released by homogenization (Mackie
et al., 1983). Twenty millilitres of 0.15 M NaCl were added to the equivalent of 0.5±2 g
bag residue DM and the residues were then homogenized (1 min at 20 000 rpm Ultra-
264
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
Turrax, IKA-WERK, Germany). The homogenate was strained through cheesecloth and
the released microorganisms were separated by differential centrifugation as described
above.
On day 26 commencing at 0900 h samples of rumen ¯uid were obtained six times at
90 min intervals as described above. pH was determined immediately using a pH meter
(Orion Research, model 701, USA).
2.2. Laboratory analysis
DM content of feed samples was determined by drying at 1008C, while organic matter
was determined by ignition at 5508C for 6 h. The N content of feeds and of residues
remaining in synthetic ®bre bags following soaking or incubation were determined by a
Kjeldahl procedure (AOAC, 1970). The enrichment of N was determined following
Kjeldahl digestion of bag residues and isolated microbe-rich fractions, distillation of
these digests or of rumen ¯uid to isolate ammonium sulphate, followed by analysis using
a mass spectrometer (Sira-10, V.G. Isogas) (Nolan, 1972; Dixon and Nolan, 1986).
2.3. Calculations and statistical analyses
The abundance of 15N in samples obtained before (15NH4)2SO4 intraruminal infusions
commenced was subtracted from the abundance measured during the infusions to
calculate the enrichment of samples during the 15N infusions. The rate of irreversible loss
of the rumen ammonia pool was calculated as described by White et al. (1969). The
proportion of a microbial pool derived from the rumen ammonia pool (i.e. the transfer
quotient) was calculated as the ratio of the 15N enrichments of the respective microbial
fraction and rumen ammonia (Nolan and Leng, 1974).
Data were analysed as a split-plot analysis of variance using GENSTAT (Version 5,
release 3.22) where sheep were considered in the main plot and the measurements within
sheep (hays incubated in the synthetic ®bre bags, time of incubation and their interactions
with diet and each other) in the sub plot. Planned comparisons between means were based
on least signi®cant differences (l.s.d.) used when the F-test was signi®cant. The planned
comparisons were between hays incubated in the synthetic ®bre bags within incubation
times, within diets when the diet hay interaction was signi®cant, and between
incubation times within hays.
3. Results
3.1. Sheep, feeds and rumen ammonia
The sheep readily consumed all of the allocated rations and were in good health
throughout the experiment. The composition of the feedstuffs fed to the sheep and
incubated in synthetic ®bre bags in the rumen are given in Table 1. N content of the
feedstuffs incubated in the synthetic ®bre bags was high for the lucerne hay (34.6 g N/kg
DM) and low for the oat hay (9.0 g N/kg DM) and barley straw (5.1 g N/kg DM).
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
265
Table 1
Composition (g/kg) of feedstuffs fed to the sheep or incubated in synthetic ®bre bags in the rumena
Feedstuff
Dry matter
Diet components
Oat hay
892
Lucerne hay
882
Barley grain
874
Forages incubated in synthetic ®bre bags
Barley straw
907
Oat hay
899
Lucerne hay
879
Organic matterb
Total nitrogenb
Insoluble nitrogenc
925
907
976
9.0
26.9
20.2
±
±
±
947
916
890
5.1
9.0
34.6
4.1
5.0
36.2
a
Insoluble nitrogen content was determined by soaking synthetic ®bre bags containing the hay in 0.15 M
NaCl before washing and drying.
b
Dry matter basis.
c
Insoluble nitrogen per unit of insoluble dry matter.
Intakes of total DM (860 and 867 g DM/day for diets 1 and 2, respectively) and of total
N (15.4 and 16.5 g N/day) were similar for the two diets. Also, rumen ¯uid pH (pH 6.63
and pH 6.46), concentration of ammonia in rumen ¯uid (113 and 136 mg N/l) and the rate
of irreversible loss of the rumen ammonia pool (10.5 and 11.9 g N/day) did not differ
(p > 0.05) between diets. The coef®cient of variation within sheep for the enrichment of
rumen ammonia ranged from 14 to 23% (average 20%), while that of the enrichment of
microbial N isolated from strained rumen ¯uid ranged from 3 to 6% (average 4%).
3.2. DM disappearance from synthetic ®bre bags
DM disappearance from the synthetic ®bre bags of 44 mm pore size is shown in Table 2.
DM disappearance due to soaking bags in saline before washing was 100, 313 and
373 mg/g DM for barley straw, oat hay and lucerne hay, respectively. There was a
signi®cant (p < 0.05) (diet time of incubation type of hay incubated) interaction
effect on DM disappearance. DM disappearance of barley straw was lower (p < 0.01) than
that of oat hay, which was lower (p < 0.001) than that of lucerne hay, after both 6 and 24 h
incubation. After 24 h incubation the DM disappearance of barley straw was reduced
(p < 0.01) by 21% (from 515 to 408 mg/g) due to including barley grain in the diet, and
disappearance of oat hay was reduced (p < 0.05) by 13% (from 606 to 526 mg/g). DM
disappearance of lucerne hay was not different (p > 0.05) between the two diets after 24 h
incubation (809 and 783 mg/g).
3.3. Total N and microbial N contents of synthetic ®bre bag residues
For each hay the percentage of adherent microbial N associated with bag residues
derived from rumen ammonia (the transfer quotient), was not affected by diet (p > 0.05).
However, the transfer quotient was in¯uenced by the type of hay (p < 0.001) and the
incubation time (p < 0.001), and there was an interaction (p < 0.05) between type of hay
and incubation time (Table 2). Averaged across the two diets, this transfer quotient was
266
Measurement
Diet 1
Diet 2
Diet
Hay
Diet hay
Time
Diet time
Hay time
Diet hay time
*
a
barley straw
oat hay
lucerne hay
barley straw
oat hay
lucerne hay
probability
s.e.d.
probability
s.e.d.
probability
s.e.d.
probability
s.e.d.
probability
s.e.d.
probability
s.e.d.
probability
s.e.d.
p < 0.05; **p < 0.01; ***p < 0.001.
Not signi®cant.
DM disappearance
from bags (mg/g
DM)
Transfer quotient (adherent
microbial N derived from
rumen NH3) (%)
Bag residue N
(mg total N/g
DM)
Proportion of bag residue
total N consisting of
microbial N (%)
Bag microbial N
(mg N/g residue
DM)
6h
24 h
6h
24 h
6h
24 h
6h
24 h
6h
24 h
180
398
706
151
358
640
515
606
809
408
526
783
49
40
24
45
48
27
64
74
29
89
86
37
5.3
7.1
4.8
5.9
11.7
8.8
5.3
7.9
4.8
7.0
10.7
10.1
n.s.
0.49
***
0.56
n.s.
0.81
n.s.
0.46
n.s.
0.67
**
0.79
n.s.
1.13
82
76
47
67
77
60
81
74
61
82
88
76
4.4
3.7
4.9
3.6
3.8
6.0
5.5
4.3
5.4
6.4
6.2
7.6
*
21.3
***
6.7
n.s.
22.7
***
5.4
*
22.0
***
9.4
***
24.6
n.s.a
8.1
***
4.8
n.s.
9.8
***
3.9
n.s.
9.0
*
6.8
n.s.
11.9
n.s.
4.6
**
5.3
n.s.
7.7
n.s.
4.3
n.s.
6.3
n.s.
7.5
n.s.
10.7
*
0.30
***
0.34
n.s.
0.49
***
0.28
*
0.41
n.s.
0.49
n.s.
0.69
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
Table 2
Measurements of disappearance of dry matter (DM), the transfer quotient representing the proportion of attached microbial N derived from the rumen NH3-N pool, the
content of total N and microbial N in bag residues and the proportion of this residue N consisting of microbial N determined using synthetic ®bre bags incubated in the
rumen and 15N tracers
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
267
low for lucerne hay (26 and 33% at 6 and 24 h, respectively), greater (p < 0.05 and
p < 0.01) for oat hay and barley straw after 6 h (44 and 47%, respectively), and even
greater (p < 0.01) after 24 h incubation of oat hay and barley straw (80 and 77%,
respectively). Microorganisms isolated from strained rumen ¯uid represented a different
kinetic pool to the microorganisms associated with the synthetic ®bre bag residues. Thus,
the transfer quotient for the N in the free-¯oating microbial pool derived from rumen
ammonia could not be compared directly with the transfer quotients for the attached
microbial N in bag residues after incubation for 6 or 24 h; the latter represent the pools of
microbial N attached to feed ingested 6 or 24 h previously in these continuously fed
sheep. The transfer quotient for the proportion of microbial N in the pool of free-¯oating
microorganisms derived from rumen ammonia was on average 57% and was not different
between diets. This indicated that the contribution of the rumen ammonia pool as
substrate for microorganisms in strained rumen ¯uid was intermediate between the values
for oat hay and lucerne hay incubated in the synthetic ®bre bags for the de®ned intervals.
The proportion of total N in the bag residues consisting of microbial N was not affected
(p > 0.05) by diet, but was in¯uenced by type of hay in the bags (p < 0.01) and was
increased (p 0.05) by the longer incubation time (Table 2). This proportion was lower
(p < 0.01) in residues of lucerne hay (54 and 69% at 6 and 24 h, respectively) than of oat
hay or barley straw (75±76% and 81% at 6 and 24 h, respectively).
Total N content of the bag residues was not affected by diet (p > 0.05), but there was a
signi®cant (p < 0.01) interaction between incubation time and type of hay (Table 2). As
incubation time increased from 6 to 24 h the N content of barley straw bag residues
increased (p < 0.01) from 5.3 to 7.5 mg N/g residue DM, and the N content of oat hay bag
residues increased (p < 0.05) from 4.8 to 6.5 mg N/g residue DM. However, N content of
lucerne hay bag residues was reduced (p < 0.05) during the same interval from 11.2 to
9.4 mg N/g residue DM. There was a signi®cant (p < 0.05) (diet time of incubation)
interaction effect on the content of microbial N in the bag residues. This content was not
affected (p > 0.05) by diet after 6 h incubation (4.3 and 4.4 mg N/g residue DM for diets 1
and 2, respectively), and was similar after 24 h incubation in sheep fed diet 1 (5.1 mg N/g
residue DM). However, the microbial N content in bag residues was increased (p < 0.01)
in sheep fed diet 2 after 24 h incubation (6.7 mg N/g residue DM). In addition, the
microbial N content of barley straw and oat hay bag residues (on average across diets 5.0
and 4.5 mg N/g residue DM) were similar, and were lower (p < 0.01) than for lucerne hay
bag residues (6.0 mg N/g residue DM).
4. Discussion
The present experiment showed that the microorganisms adherent to forages, and
particularly to the high-protein lucerne hay, obtained much of their N substrates from the
plant material rather than from the rumen ammonia pool even though ammonia
concentrations in rumen ¯uid (113 and 136 mg N/l in the 2 diets) were likely to be
adequate for microbial activity (Satter and Slyter, 1974; Morrison and Mackie, 1996).
Numerous studies (Akin and Barton, 1983; Cheng et al., 1984; Orpin, 1984) have shown
that it is the rumen microorganisms in close physical association with plant material
268
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
which are primarily responsible for the digestion of ®brous material. Where there is such
close physical association, it is perhaps not surprising that microorganisms should utilize
preferentially substrates which are available in the forage. The form of the N substrate
used by the microorganisms was not determined in the present experiment. Although the
principal N substrate used by ®brolytic microorganisms is usually ammonia (Leng and
Nolan, 1984; Morrison and Mackie, 1996), preferential use of N in the form of peptides
and amino acids has been shown when these are available (Maeng et al., 1976).
Alternatively ammonia produced by degradation of plant protein may have been utilized
without mixing with the rumen ammonia pool. Regardless of the form of the N substrate,
it was clear that in the present study the microorganisms digesting the high protein forage
were largely independent of the rumen ammonia pool, but that those digesting the barley
straw and the oat hay were more dependent on the rumen ammonia pool, particularly
when digestion had been proceeding for 24 h. Presumably, the greater dependence after
24 h than 6 h digestion was because much of the N that was in the plant material was
utilized early in the digestion process, leaving the rumen ammonia pool as the principal
source of N substrate. This is consistent with observations that microbial digestion of
high-protein forages is less adversely affected than that of low protein forages by low
rumen ammonia concentrations (Dixon, 1999), and that higher rumen ammonia
concentrations are needed for maximum digestion rates of low N forages (Alvarez
et al., 1984; Krebs and Leng, 1984; Morrison and Mackie, 1996). In addition,
measurements in a number of studies of the proportion of microbial N in strained rumen
¯uid apparently derived from the rumen ammonia pool during intraruminal 15NH4
infusions provide further evidence that the contribution of rumen ammonia to microbial N
varies among forages. Although with diets based on low N forages most (0.84±0.95)
microbial N was derived from the rumen ammonia pool (Salter et al., 1979; Neutze et al.,
1986; Hennessy and Nolan, 1988), with medium to high N forage diets only 0.4±0.6 of
microbial N was derived from rumen ammonia and the remainder was derived directly
from forage N (Nolan et al., 1976; Kennedy and Milligan, 1978; Nolan and Stachiw,
1979; Dixon and Nolan, 1986).
An assumption in the present study was the microorganisms released by homogenisation were representative in 15N enrichment to the groups of microorganisms adherent to
the forage residues in the synthetic ®bre bags. In other studies only 30±60% of the
microbes adherent to plant fragments were released by similar homogenisation (Kennedy
et al., 1984; Whitehouse et al., 1994) or by chilling and washing procedures (Craig et al.,
1987). Any difference in 15N enrichment between the microorganisms released and those
remaining bound to the plant fragments would have introduced error into the estimates
both of the N substrate derived from the rumen ammonia pool and the amount of
microbial N remaining in the bag residues. We speculate that the groups of
microorganisms not released by homogenisation were those most intimately associated
with the plant fragments, and that such groups were most likely to obtain substrate N
directly from the plant material. Thus, if error did occur for this reason it is likely to have
resulted in overestimation of the contribution of the rumen ammonia to adherent
microbial N and underestimation of the microbial N content of the bag residues.
DM disappearance from the synthetic ®bre bags indicated that digestion of hay,
particularly of the barley straw and the oat hay, was reduced by inclusion of the barley
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
269
grain in the diet. Depression of forage digestion in the rumen has often been reported in
response to inclusion of cereal grain in the diet, even when rumen pH is maintained at
levels of pH 6.4 or above as in the present experiment (Mould et al., 1984; Dixon and
Stockdale, 1999). Also, the greater depression of barley straw and of oat hay than of
lucerne hay is in agreement with previous reports of an inverse relationship between
forage digestibility and the depression in digestion due to dietary concentrates (Dixon and
Stockdale, 1999).
The bags of large 100 mm pore size were used for the measurements of microbial
colonization to increase the likelihood that the microbial populations inside the bags
would be similar to those in rumen digesta. Bacterial and protozoal populations inside
synthetic ®bre bags may differ to those of rumen digesta (Meyer and Mackie, 1986).
Although differences were exacerbated when the pore size of bags was
83 (2000) 261±272
Colonization and source of N substrates used by
microorganisms digesting forages incubated in
synthetic ®bre bags in the rumen
R.M. Dixon*,1, S. Chanchai2
School of Agriculture and Forestry, The University of Melbourne, Parkville, Vic 3052, Australia
Received 13 April 1999; received in revised form 5 October 1999; accepted 11 November 1999
Abstract
Six mature sheep were fed restricted amounts of either a medium quality roughage or a 1 : 1
mixture of the roughage and barley grain. Disappearance of DM of three roughages (barley straw,
oat hay and lucerne hay) from synthetic ®bre bags incubated in the rumen for 6 and 24 h was
determined. Also, during intraruminal infusions of 15NH4C1, synthetic ®bre bags containing each of
the three roughages were incubated in the rumen for 6 and 24 h. The origins and amounts of
adherent microbial N associated with the bag residues after incubation and washing were estimated
from the 15N enrichments of rumen ammonia, adherent microbial N and bag residue total N. The
proportion of adherent microbial N derived from the rumen ammonia pool was not affected
(p > 0.05) by diet, but was lower (p < 0.05 or p < 0.01) for microorganisms adherent to lucerne hay
bag residues (26 and 33% at 6 and 24 h, respectively) than microorganisms adherent to barley straw
(47 and 77% at 6 and 24 h, respectively) or oat hay bag residues (44 and 80% at 6 and 24 h,
respectively). The proportion of bag residue N consisting of microbial N was not affected (p > 0.05)
by the diet, but was lower (p < 0.01) in lucerne hay bag residues (54 and 69% at 6 and 24 h,
respectively) than in barley straw or oat hay bag residues (75±76% at 6 h and 81% at 24 h).
Microbial N remaining associated with bag residues ranged from 3.7 to 7.6 mg microbial N/g
residual DM. Because of this microbial N associated with bag residues, rumen degradability of
lucerne hay N was underestimated by ca. 12 and 4% at 6 and 24 h, respectively. The
underestimation of the rumen degradability of oat hay N was more than 26% units, and that of
barley straw N was more than 75% units. In conclusion, this experiment indicated that the
microorganisms digesting low N forages are much more dependent on rumen ammonia as a N
*
Corresponding author. Tel.: 61-747-849170; fax: 61-747-849232.
E-mail address: dixonr@prose.dpi.qld.gov.au (R.M. Dixon).
1
Present address: Queensland Beef Industry Institute, Swan's Lagoon Research Station, Millaroo, Ayr, Qld
4807.
2
Present address: Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand.
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 1 2 8 - 5
262
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
substrate than those digesting high N forages, and that microbial N associated with the residues
remaining in synthetic ®bre bag residues following incubation and washing was substantial.
# 2000 Elsevier Science B.V. All rights reserved.
Keywords: Sheep; Synthetic ®bre bags; Microbial colonization
1. Introduction
The nitrogen (N) requirements of ruminants are usually considered in terms of the
nitrogenous substrates for rumen microorganisms (rumen degradable protein (RDP) or
effective rumen degradable protein (ERDP)), and dietary protein escaping rumen
fermentation but digested in the small intestine (SCA, 1990; AFRC, 1993). Since most
ERDP is utilised as a microbial substrate after its conversion to ammonia (Leng and
Nolan, 1984; Morrison and Mackie, 1996), an understanding of the requirements of
microorganisms for ammonia N to ferment various classes of feedstuffs is essential for
understanding dietary requirements for ERDP. Current feeding standards assume that the
amount of ERDP required is, with some corrections, equivalent to the amount of net
microbial protein synthesised and that the latter is a function of the availability of
fermentable energy in the rumen. However, the concentrations of rumen ammonia
required for the maximum rate of fermentation and voluntary intake of ®brous feeds, and
the adverse effects of low concentrations, appear to be greater for low quality forages
(Alvarez et al., 1984; Krebs and Leng, 1984; Perdok et al., 1988). In addition, the extent
to which microorganisms digesting forages depend on rumen ammonia as a N substrate
appears to vary with the protein content of the forage (Nolan et al., 1976; Neutze et al.,
1986; Dixon, 1999).
Disappearance of the nitrogenous components of feedstuffs from synthetic ®bre bags
incubated in the rumen has been accepted as a standard procedure to evaluate the N
degradability and ERDP content of feedstuffs (SCA, 1990; AFRC, 1993). However, any
microbial N remaining in synthetic ®bre bag residues after washing results in
underestimation of the degradability of the dietary protein. The underestimation is small
for protein meals (Mathers and Aitchison, 1981; Varvikko and Lindberg, 1985; Gonzalez
et al., 1998) but a number of experiments indicate that it is substantial for forages.
Microbial N remaining in residues from forages incubated in synthetic ®bre bags has
been measured using 35S tracers (Mathers and Aitchison, 1981; Kennedy et al., 1984),
diaminopimelic acid (Nocek and Grant, 1987; Olubobokun et al., 1990; Alexandrov,
1998), D-amino acids (Rooke et al., 1984), or by dilution of 15N tracer incorporated into
the plant material (Varvikko and Lindberg, 1985; Wanderley et al., 1993). However, there
may be substantial inaccuracies in these estimates due to dif®culties with methods used to
measure the microbial N in synthetic ®bre bag residues.
The present experiment investigated the extent to which adherent microorganisms
digesting forages ranging in N content utilized the rumen ammonia pool as a N substrate.
A second objective was to measure microbial N remaining in residues of forages
incubated in synthetic ®bre bags using an 15N tracer procedure which avoided some of the
dif®culties associated with the procedures used in previous experiments. Measurements
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
263
were made for three hays ranging in N content and in sheep fed a forage or a mixed
forage±cereal grain diet.
2. Materials and methods
2.1. Sheep,
15
NH4Cl infusions and synthetic ®bre bags
Six mature Merino sheep (liveweight mean 48, range 43±53 kg; 2±3 years of age)
surgically prepared with rumen cannulas were held indoors in metabolism crates. Three
sheep were allocated at random to each of two diets consisting, on an air dry basis, of
500 g chopped oat hay and 500 g chopped lucerne hay (Diet 1), or 250 g chopped oat hay,
250 g chopped lucerne hay and 500 g whole barley grain (Diet 2). From day 7 the diets
were fed from continuously moving belts and the room was continuously lit.
On days 16 and 18 duplicate synthetic ®bre bags containing barley straw, oat hay or
lucerne hay were inserted into the rumen at 0900 h and removed after 6 and 24 h. The
bags (50 120 mm, 44 mm pore size mono®lament nylon cloth (Swiss Screens,
Moorabbin, Victoria, Australia)) contained ca. 2.0, 2.3 and 2.5 g of barley straw, oat
hay and lucerne hay, respectively, and also a 3.5 g metal ball as a weight. These hays were
ground through a 1 mm screen (Makla mill, Crompton Parkinson model 8302A-P,
Australia). On removal from the rumen bags were brie¯y washed and then stored at 58C
until all bags had been removed. The bags were then washed manually under running
water until the washings were clear and dried at 708C. The solubility of dry matter (DM)
of the hays was also determined by soaking ®ve bags containing each ground hay in
0.15 M NaCl for 3 h before washing and drying.
Intra-ruminal infusions of 15NH4C1 (30 mg 15N in 850 ml aqueous solution per day,
98.9 atom% excess, Amersham International, UK) were commenced at 0900 h on day 22
and were continued for 96 h. Samples of rumen ¯uid (50 ml) were obtained by suction
through a gauze covered cage suspended in the ventral sac of the rumen before infusion of
15
N tracer commenced and also six times from 48 to 96 h of the infusions. The samples
were acidi®ed (0.5 ml 5 M H2SO4) and were then centrifuged (1000 g for 1 min). The
supernatant thus obtained was again centrifuged (14 000 g for 15 min) to separate
rumen ¯uid free of particulate matter and a microbe-rich fraction (Nolan, 1972). The
microbe-rich fraction was washed once with 0.15 M NaCl, isolated again by
centrifugation (14 000 g for 15 min) and then stored frozen pending 15N analysis.
On days 24 and 25, during the intra-ruminal 15NH4Cl infusions, synthetic ®bre bags
containing the barley straw, oat hay and lucerne hay were inserted at 0900 h and were
removed after 6 and 24 h. The procedures for these synthetic ®bre bags were as described
above, except that the bags contained 4.0, 4.6 and 5.0 g barley straw, oat hay and lucerne
hay, respectively, and the bags (85 160 mm) were made from a 100 mm pore size
mono®lament nylon cloth (Swiss Screens, Moorabbin, Victoria, Australia). Following
washing the bags were stored at 58C. Microorganisms adherent to the residues remaining
in the bags following incubation and washing were released by homogenization (Mackie
et al., 1983). Twenty millilitres of 0.15 M NaCl were added to the equivalent of 0.5±2 g
bag residue DM and the residues were then homogenized (1 min at 20 000 rpm Ultra-
264
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
Turrax, IKA-WERK, Germany). The homogenate was strained through cheesecloth and
the released microorganisms were separated by differential centrifugation as described
above.
On day 26 commencing at 0900 h samples of rumen ¯uid were obtained six times at
90 min intervals as described above. pH was determined immediately using a pH meter
(Orion Research, model 701, USA).
2.2. Laboratory analysis
DM content of feed samples was determined by drying at 1008C, while organic matter
was determined by ignition at 5508C for 6 h. The N content of feeds and of residues
remaining in synthetic ®bre bags following soaking or incubation were determined by a
Kjeldahl procedure (AOAC, 1970). The enrichment of N was determined following
Kjeldahl digestion of bag residues and isolated microbe-rich fractions, distillation of
these digests or of rumen ¯uid to isolate ammonium sulphate, followed by analysis using
a mass spectrometer (Sira-10, V.G. Isogas) (Nolan, 1972; Dixon and Nolan, 1986).
2.3. Calculations and statistical analyses
The abundance of 15N in samples obtained before (15NH4)2SO4 intraruminal infusions
commenced was subtracted from the abundance measured during the infusions to
calculate the enrichment of samples during the 15N infusions. The rate of irreversible loss
of the rumen ammonia pool was calculated as described by White et al. (1969). The
proportion of a microbial pool derived from the rumen ammonia pool (i.e. the transfer
quotient) was calculated as the ratio of the 15N enrichments of the respective microbial
fraction and rumen ammonia (Nolan and Leng, 1974).
Data were analysed as a split-plot analysis of variance using GENSTAT (Version 5,
release 3.22) where sheep were considered in the main plot and the measurements within
sheep (hays incubated in the synthetic ®bre bags, time of incubation and their interactions
with diet and each other) in the sub plot. Planned comparisons between means were based
on least signi®cant differences (l.s.d.) used when the F-test was signi®cant. The planned
comparisons were between hays incubated in the synthetic ®bre bags within incubation
times, within diets when the diet hay interaction was signi®cant, and between
incubation times within hays.
3. Results
3.1. Sheep, feeds and rumen ammonia
The sheep readily consumed all of the allocated rations and were in good health
throughout the experiment. The composition of the feedstuffs fed to the sheep and
incubated in synthetic ®bre bags in the rumen are given in Table 1. N content of the
feedstuffs incubated in the synthetic ®bre bags was high for the lucerne hay (34.6 g N/kg
DM) and low for the oat hay (9.0 g N/kg DM) and barley straw (5.1 g N/kg DM).
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
265
Table 1
Composition (g/kg) of feedstuffs fed to the sheep or incubated in synthetic ®bre bags in the rumena
Feedstuff
Dry matter
Diet components
Oat hay
892
Lucerne hay
882
Barley grain
874
Forages incubated in synthetic ®bre bags
Barley straw
907
Oat hay
899
Lucerne hay
879
Organic matterb
Total nitrogenb
Insoluble nitrogenc
925
907
976
9.0
26.9
20.2
±
±
±
947
916
890
5.1
9.0
34.6
4.1
5.0
36.2
a
Insoluble nitrogen content was determined by soaking synthetic ®bre bags containing the hay in 0.15 M
NaCl before washing and drying.
b
Dry matter basis.
c
Insoluble nitrogen per unit of insoluble dry matter.
Intakes of total DM (860 and 867 g DM/day for diets 1 and 2, respectively) and of total
N (15.4 and 16.5 g N/day) were similar for the two diets. Also, rumen ¯uid pH (pH 6.63
and pH 6.46), concentration of ammonia in rumen ¯uid (113 and 136 mg N/l) and the rate
of irreversible loss of the rumen ammonia pool (10.5 and 11.9 g N/day) did not differ
(p > 0.05) between diets. The coef®cient of variation within sheep for the enrichment of
rumen ammonia ranged from 14 to 23% (average 20%), while that of the enrichment of
microbial N isolated from strained rumen ¯uid ranged from 3 to 6% (average 4%).
3.2. DM disappearance from synthetic ®bre bags
DM disappearance from the synthetic ®bre bags of 44 mm pore size is shown in Table 2.
DM disappearance due to soaking bags in saline before washing was 100, 313 and
373 mg/g DM for barley straw, oat hay and lucerne hay, respectively. There was a
signi®cant (p < 0.05) (diet time of incubation type of hay incubated) interaction
effect on DM disappearance. DM disappearance of barley straw was lower (p < 0.01) than
that of oat hay, which was lower (p < 0.001) than that of lucerne hay, after both 6 and 24 h
incubation. After 24 h incubation the DM disappearance of barley straw was reduced
(p < 0.01) by 21% (from 515 to 408 mg/g) due to including barley grain in the diet, and
disappearance of oat hay was reduced (p < 0.05) by 13% (from 606 to 526 mg/g). DM
disappearance of lucerne hay was not different (p > 0.05) between the two diets after 24 h
incubation (809 and 783 mg/g).
3.3. Total N and microbial N contents of synthetic ®bre bag residues
For each hay the percentage of adherent microbial N associated with bag residues
derived from rumen ammonia (the transfer quotient), was not affected by diet (p > 0.05).
However, the transfer quotient was in¯uenced by the type of hay (p < 0.001) and the
incubation time (p < 0.001), and there was an interaction (p < 0.05) between type of hay
and incubation time (Table 2). Averaged across the two diets, this transfer quotient was
266
Measurement
Diet 1
Diet 2
Diet
Hay
Diet hay
Time
Diet time
Hay time
Diet hay time
*
a
barley straw
oat hay
lucerne hay
barley straw
oat hay
lucerne hay
probability
s.e.d.
probability
s.e.d.
probability
s.e.d.
probability
s.e.d.
probability
s.e.d.
probability
s.e.d.
probability
s.e.d.
p < 0.05; **p < 0.01; ***p < 0.001.
Not signi®cant.
DM disappearance
from bags (mg/g
DM)
Transfer quotient (adherent
microbial N derived from
rumen NH3) (%)
Bag residue N
(mg total N/g
DM)
Proportion of bag residue
total N consisting of
microbial N (%)
Bag microbial N
(mg N/g residue
DM)
6h
24 h
6h
24 h
6h
24 h
6h
24 h
6h
24 h
180
398
706
151
358
640
515
606
809
408
526
783
49
40
24
45
48
27
64
74
29
89
86
37
5.3
7.1
4.8
5.9
11.7
8.8
5.3
7.9
4.8
7.0
10.7
10.1
n.s.
0.49
***
0.56
n.s.
0.81
n.s.
0.46
n.s.
0.67
**
0.79
n.s.
1.13
82
76
47
67
77
60
81
74
61
82
88
76
4.4
3.7
4.9
3.6
3.8
6.0
5.5
4.3
5.4
6.4
6.2
7.6
*
21.3
***
6.7
n.s.
22.7
***
5.4
*
22.0
***
9.4
***
24.6
n.s.a
8.1
***
4.8
n.s.
9.8
***
3.9
n.s.
9.0
*
6.8
n.s.
11.9
n.s.
4.6
**
5.3
n.s.
7.7
n.s.
4.3
n.s.
6.3
n.s.
7.5
n.s.
10.7
*
0.30
***
0.34
n.s.
0.49
***
0.28
*
0.41
n.s.
0.49
n.s.
0.69
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
Table 2
Measurements of disappearance of dry matter (DM), the transfer quotient representing the proportion of attached microbial N derived from the rumen NH3-N pool, the
content of total N and microbial N in bag residues and the proportion of this residue N consisting of microbial N determined using synthetic ®bre bags incubated in the
rumen and 15N tracers
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
267
low for lucerne hay (26 and 33% at 6 and 24 h, respectively), greater (p < 0.05 and
p < 0.01) for oat hay and barley straw after 6 h (44 and 47%, respectively), and even
greater (p < 0.01) after 24 h incubation of oat hay and barley straw (80 and 77%,
respectively). Microorganisms isolated from strained rumen ¯uid represented a different
kinetic pool to the microorganisms associated with the synthetic ®bre bag residues. Thus,
the transfer quotient for the N in the free-¯oating microbial pool derived from rumen
ammonia could not be compared directly with the transfer quotients for the attached
microbial N in bag residues after incubation for 6 or 24 h; the latter represent the pools of
microbial N attached to feed ingested 6 or 24 h previously in these continuously fed
sheep. The transfer quotient for the proportion of microbial N in the pool of free-¯oating
microorganisms derived from rumen ammonia was on average 57% and was not different
between diets. This indicated that the contribution of the rumen ammonia pool as
substrate for microorganisms in strained rumen ¯uid was intermediate between the values
for oat hay and lucerne hay incubated in the synthetic ®bre bags for the de®ned intervals.
The proportion of total N in the bag residues consisting of microbial N was not affected
(p > 0.05) by diet, but was in¯uenced by type of hay in the bags (p < 0.01) and was
increased (p 0.05) by the longer incubation time (Table 2). This proportion was lower
(p < 0.01) in residues of lucerne hay (54 and 69% at 6 and 24 h, respectively) than of oat
hay or barley straw (75±76% and 81% at 6 and 24 h, respectively).
Total N content of the bag residues was not affected by diet (p > 0.05), but there was a
signi®cant (p < 0.01) interaction between incubation time and type of hay (Table 2). As
incubation time increased from 6 to 24 h the N content of barley straw bag residues
increased (p < 0.01) from 5.3 to 7.5 mg N/g residue DM, and the N content of oat hay bag
residues increased (p < 0.05) from 4.8 to 6.5 mg N/g residue DM. However, N content of
lucerne hay bag residues was reduced (p < 0.05) during the same interval from 11.2 to
9.4 mg N/g residue DM. There was a signi®cant (p < 0.05) (diet time of incubation)
interaction effect on the content of microbial N in the bag residues. This content was not
affected (p > 0.05) by diet after 6 h incubation (4.3 and 4.4 mg N/g residue DM for diets 1
and 2, respectively), and was similar after 24 h incubation in sheep fed diet 1 (5.1 mg N/g
residue DM). However, the microbial N content in bag residues was increased (p < 0.01)
in sheep fed diet 2 after 24 h incubation (6.7 mg N/g residue DM). In addition, the
microbial N content of barley straw and oat hay bag residues (on average across diets 5.0
and 4.5 mg N/g residue DM) were similar, and were lower (p < 0.01) than for lucerne hay
bag residues (6.0 mg N/g residue DM).
4. Discussion
The present experiment showed that the microorganisms adherent to forages, and
particularly to the high-protein lucerne hay, obtained much of their N substrates from the
plant material rather than from the rumen ammonia pool even though ammonia
concentrations in rumen ¯uid (113 and 136 mg N/l in the 2 diets) were likely to be
adequate for microbial activity (Satter and Slyter, 1974; Morrison and Mackie, 1996).
Numerous studies (Akin and Barton, 1983; Cheng et al., 1984; Orpin, 1984) have shown
that it is the rumen microorganisms in close physical association with plant material
268
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
which are primarily responsible for the digestion of ®brous material. Where there is such
close physical association, it is perhaps not surprising that microorganisms should utilize
preferentially substrates which are available in the forage. The form of the N substrate
used by the microorganisms was not determined in the present experiment. Although the
principal N substrate used by ®brolytic microorganisms is usually ammonia (Leng and
Nolan, 1984; Morrison and Mackie, 1996), preferential use of N in the form of peptides
and amino acids has been shown when these are available (Maeng et al., 1976).
Alternatively ammonia produced by degradation of plant protein may have been utilized
without mixing with the rumen ammonia pool. Regardless of the form of the N substrate,
it was clear that in the present study the microorganisms digesting the high protein forage
were largely independent of the rumen ammonia pool, but that those digesting the barley
straw and the oat hay were more dependent on the rumen ammonia pool, particularly
when digestion had been proceeding for 24 h. Presumably, the greater dependence after
24 h than 6 h digestion was because much of the N that was in the plant material was
utilized early in the digestion process, leaving the rumen ammonia pool as the principal
source of N substrate. This is consistent with observations that microbial digestion of
high-protein forages is less adversely affected than that of low protein forages by low
rumen ammonia concentrations (Dixon, 1999), and that higher rumen ammonia
concentrations are needed for maximum digestion rates of low N forages (Alvarez
et al., 1984; Krebs and Leng, 1984; Morrison and Mackie, 1996). In addition,
measurements in a number of studies of the proportion of microbial N in strained rumen
¯uid apparently derived from the rumen ammonia pool during intraruminal 15NH4
infusions provide further evidence that the contribution of rumen ammonia to microbial N
varies among forages. Although with diets based on low N forages most (0.84±0.95)
microbial N was derived from the rumen ammonia pool (Salter et al., 1979; Neutze et al.,
1986; Hennessy and Nolan, 1988), with medium to high N forage diets only 0.4±0.6 of
microbial N was derived from rumen ammonia and the remainder was derived directly
from forage N (Nolan et al., 1976; Kennedy and Milligan, 1978; Nolan and Stachiw,
1979; Dixon and Nolan, 1986).
An assumption in the present study was the microorganisms released by homogenisation were representative in 15N enrichment to the groups of microorganisms adherent to
the forage residues in the synthetic ®bre bags. In other studies only 30±60% of the
microbes adherent to plant fragments were released by similar homogenisation (Kennedy
et al., 1984; Whitehouse et al., 1994) or by chilling and washing procedures (Craig et al.,
1987). Any difference in 15N enrichment between the microorganisms released and those
remaining bound to the plant fragments would have introduced error into the estimates
both of the N substrate derived from the rumen ammonia pool and the amount of
microbial N remaining in the bag residues. We speculate that the groups of
microorganisms not released by homogenisation were those most intimately associated
with the plant fragments, and that such groups were most likely to obtain substrate N
directly from the plant material. Thus, if error did occur for this reason it is likely to have
resulted in overestimation of the contribution of the rumen ammonia to adherent
microbial N and underestimation of the microbial N content of the bag residues.
DM disappearance from the synthetic ®bre bags indicated that digestion of hay,
particularly of the barley straw and the oat hay, was reduced by inclusion of the barley
R.M. Dixon, S. Chanchai / Animal Feed Science and Technology 83 (2000) 261±272
269
grain in the diet. Depression of forage digestion in the rumen has often been reported in
response to inclusion of cereal grain in the diet, even when rumen pH is maintained at
levels of pH 6.4 or above as in the present experiment (Mould et al., 1984; Dixon and
Stockdale, 1999). Also, the greater depression of barley straw and of oat hay than of
lucerne hay is in agreement with previous reports of an inverse relationship between
forage digestibility and the depression in digestion due to dietary concentrates (Dixon and
Stockdale, 1999).
The bags of large 100 mm pore size were used for the measurements of microbial
colonization to increase the likelihood that the microbial populations inside the bags
would be similar to those in rumen digesta. Bacterial and protozoal populations inside
synthetic ®bre bags may differ to those of rumen digesta (Meyer and Mackie, 1986).
Although differences were exacerbated when the pore size of bags was