Colonization and source of N substrates used by
microorganisms digesting forages incubated in
synthetic ®bre bags in the rumen
R.M. Dixon
*,1, S. Chanchai
2School 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 of15NH4C1, 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 the15N 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 orp< 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
83 (2000) 261±272
*Corresponding author. Tel.:61-747-849170; fax:61-747-849232.
E-mail address: dixonr@prose.dpi.qld.gov.au (R.M. Dixon).
1Present address: Queensland Beef Industry Institute, Swan's Lagoon Research Station, Millaroo, Ayr, Qld
4807.
2Present address: Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand.
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 of15N 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.
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,15NH4Cl 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 (50120 mm, 44mm 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 of15NH4C1 (30 mg15N 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 (1000gfor 1 min). The supernatant thus obtained was again centrifuged (14 000g 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 000g for 15 min) and then stored frozen pending15N analysis.
On days 24 and 25, during the intra-ruminal15NH4Cl 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 (85160 mm) were made from a 100mm 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
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 of15N in samples obtained before (15NH4)2SO4intraruminal infusions commenced was subtracted from the abundance measured during the infusions to calculate the enrichment of samples during the15N 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 the15N 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 theF-test was signi®cant. The planned comparisons were between hays incubated in the synthetic ®bre bags within incubation times, within diets when the diethay interaction was signi®cant, and between incubation times within hays.
3. Results
3.1. Sheep, feeds and rumen ammonia
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 44mm 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) (diettime of incubationtype 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 Table 1
Composition (g/kg) of feedstuffs fed to the sheep or incubated in synthetic ®bre bags in the rumena
Feedstuff Dry matter Organic matterb Total nitrogenb Insoluble nitrogenc
Diet components
Oat hay 892 925 9.0 ±
Lucerne hay 882 907 26.9 ±
Barley grain 874 976 20.2 ±
Forages incubated in synthetic ®bre bags
Barley straw 907 947 5.1 4.1
Oat hay 899 916 9.0 5.0
Lucerne hay 879 890 34.6 36.2
aInsoluble nitrogen content was determined by soaking synthetic ®bre bags containing the hay in 0.15 M
NaCl before washing and drying.
bDry matter basis.
cInsoluble nitrogen per unit of insoluble dry matter.
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 and15N tracers
Diethay probability n.s. n.s. n.s. n.s. n.s.
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 (p0.05) 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) (diettime 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
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 homogenisa-tion were representative in15N 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 in15N 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.
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 100mm 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 <20mm, even with bags made of 53mm pore size cloth the number and diversity of cellulolytic bacterial species and protozoa in rumen digesta and inside the bag may differ. DM disappearance from the 100mm pore size bags was an average 22 and 15% greater at 6 and 24 h, respectively, than the DM disappearance from the 44mm pore size bags. This difference due to bag porosity was similar to the 17% greater rate of DM disappearance from bags of 102mm than bags of 40mm pore size reported by Nocek (1985).
The ®ndings of the present experiment agree with previous reports that substantial microbial N remains associated with forage residues remaining in synthetic ®bre bags which have been incubated in the rumen and then washed. However, for several reasons we consider that the15N tracer technique used in the present experiment is more likely than the techniques used in previous studies to have provided unbiased estimates of this microbial colonization. Firstly, since 15N tracer is a constituent of microbial N, no assumptions are required about the ratio of the microbial marker to microbial N. Use of 35
S and diaminopimelic acid as microbial markers depends on measuring or assuming a ratio of marker to microbial N, and also the latter marker is not synthesised by rumen protozoa or fungi. Dif®culties and inconsistencies with these as microbial markers are well recognised (Harrison and McAllan, 1980; Siddons et al., 1982; Kennedy et al., 1984). Secondly, in the present experiment samples of adherent microorganisms were separated from the synthetic ®bre bag residues so that the proportion of bag residue N consisting of microbial N could be calculated independently for each hay, diet and incubation time. If the proportion of microbial N in the bag residue N had instead been calculated from the enrichment of microorganisms separated from strained rumen ¯uid, the content of microbial N in bag residues would have ranged from 0.7 to 2.2 of the actual estimates. Similar dif®culties are likely to also occur with diaminopimelic acid and35S as microbial markers since differences in marker: N ratios have been reported in microbial fractions separated from rumen ¯uid and particulate material (Merry and McAllan, 1983; Kennedy et al., 1984; Olubobokun et al., 1988; Legay-Carmier and Bauchart, 1989). Nevertheless, most estimates of microbial colonization of bag residues (Mathers and Aitchison, 1981; Nocek and Grant, 1987; Beckers et al., 1995; Gonzalez et al., 1998; Alexandrov, 1998) have used microorganisms separated from rumen digesta to determine the ratio of microbial marker to microbial N or DM. Thirdly, the 15N tracer technique used in the present experiment avoided likely error due to reincorporation of tracer when microbial colonization is estimated from the dilution of15N labelled plant material with
N of natural abundance (Varvikko and Lindberg, 1985; Wanderley et al., 1993). The present experiment indicates that there would have been extensive incorporation of15N tracer from the plant material into microbial N, which would have led to underestimation of the microbial colonization of the forage residues.
The amount of microbial N remaining associated with synthetic ®bre bag residues is presumably affected by many experimental variables such as the pore size of the bags, washing procedures and chilling (Dehority and Grubb, 1980), as well as differences between the microbial markers utilized. The estimates of the proportion of bag residue N consisting of microbial N in the present experiment were generally higher than those previously reported. For lucerne hay this proportion was similar to the estimates of Olubobokun et al. (1990), but higher than the estimates of Wanderley et al. (1993) and Alexandrov (1998) and much higher than the estimates of Mathers and Aitchison (1981). Similarly estimates of the proportion of oat hay and barley straw bag residue N consisting of microbial N in the present experiment were generally higher than previous estimates for grass hays and cereal straws (Varvikko and Lindberg, 1985; Olubobokun et al., 1990; Wanderley et al., 1993; Alexandrov, 1998). Assuming that similar amounts of microbial N remained in the 44 and 100mm bag residues in the present experiment, the synthetic ®bre bag technique underestimated the rumen degradability of the lucerne hay N by 12 and 4% at 6 and 24 h, respectively. The underestimation of rumen degradability of N in the lower protein hays was much greater, being 30 and 26% at 6 and 24 h, respectively, for oat hay, and 90 and 75% at 6 and 24 h, respectively, for barley straw. Thus, in conclusion the present experiment agrees with previous reports that the synthetic ®bre bag technique may provide an acceptable estimate of the N degradability in the rumen of high protein forages which are extensively digested in the rumen. Furthermore, the precision of such estimates could be improved by incorporating corrections for microbial colonization derived from measurements such as those made in the present experiment. However, large errors are likely to occur when N degradability of low protein forages is estimated with the synthetic ®bre bags.
Acknowledgements
The authors wishes to thank the International Atomic Energy Agency of the United Nations for their ®nancial support for a fellowship for one of us (S.C.) during the period when the experimental work was conducted, Mr T. Hendy for assistance with 15N analysis and Drs J.V. Nolan and S.R. McLennan for constructive comments.
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