Effect of increasing availability of water-soluble carbohydrates on in vitro rumen fermentation

Animal Feed Science and Technology 104 (2003) 59–70

Effect of increasing availability of water-soluble carbohydrates on in vitro rumen fermentation M.R.F. Lee a,∗ , R.J. Merry a , D.R. Davies a , J.M. Moorby a , M.O. Humphreys a , M.K. Theodorou a , J.C. MacRae b , N.D. Scollan a a

Institute of Grassland and Environmental Research, Aberystwyth, Wales, SY23 3EB, UK b Rowett Research Institute, Bucksburn, Aberdeen, Scotland, AB21 9SB, UK

Received 5 September 2001; received in revised form 4 October 2002; accepted 4 October 2002

Abstract The effect of adding water-soluble carbohydrate (WSC) on the microbial fermentation of fresh perennial ryegrass was examined in an in vitro RUSITEC system over two 10 day periods. Four treatment levels of WSC were used: a basal grass and a basal grass plus a sugar infusion to raise the WSC basal grass level by approximately 1.25×; 1.5× and 1.75×. The infusion was a mixture of inulin and sucrose (80:20) infused over the first 14 h of each 24 h cycle, based on a preliminary study which indicated a linear disappearance of forage WSC over this period. With increasing WSC inclusion there were linear reductions in pH and ammonia-N (NH3 N) (P = 0.001) but only a small increase in total volatile fatty acid concentration. There was also a decrease in the proportion of acetate and an increase in propionate with increasing WSC inclusion (P = 0.02 and 0.009, respectively). Microbial nitrogen production was similar for the first three treatments of Basal to Basal × 1.5 but was lower at the highest level of WSC inclusion (P < 0.001). The efficiency of microbial protein synthesis increased from Basal to Basal × 1.5 (9.9, 10.8, 12.7 g N/kg organic matter apparently digested (OMAD), respectively) but at the highest level of WSC inclusion (Basal × 1.75) there was a reduction to 7.1 g N/kg OMAD. This may have been related to the low pH values (<6.0) at certain times during incubation and/or futile bacterial energy cycles as a result of the low nitrogen concentration in the vessels. With increasing WSC inclusion there were also significant reductions in OMAD from 14.4 g per day at Basal to 12.0 g per day at Basal × 1.75.

Abbreviations: EMPS, efficiency of microbial protein synthesis; IVDMD, in vitro dry matter digestibility; LAB, liquid associated bacteria; NAN, non-ammonia nitrogen; OMAD, organic matter apparently digested; RUSITEC, rumen simulation technique; SAB, solid associated bacteria; VFA, volatile fatty acid; WSC, water-soluble carbohydrate ∗ Corresponding author. Tel.: +44-1970-823084; fax: +44-1970-828357. E-mail address: [email protected] (M.R.F. Lee). 0377-8401/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0377-8401(02)00319-X

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This study demonstrated the effect of increasing the concentration of WSC of Lolium perenne in batch culture on the rumen microbial population. Elevation of WSC caused a drop in the pH of the effluent, ammonia-N concentration and an increase in the efficiency of microbial protein synthesis, up to Basal × 1.5, however, this was due to a drop in the organic matter apparently digested. This suggests a switch in substrate specificity of the micro-organisms which may be associated with changes in the microbial population. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Water-soluble carbohydrate; Rumen; Microbial protein; Ammonia; Volatile fatty acids

1. Introduction High sugar grasses (water-soluble carbohydrate (WSC) concentration of circa 20–40% of DM) have been shown to increase animal performance, elevate lamb growth rates (Lee et al., 2001), improve milk yields (Miller et al., 1999) and increase the flow of amino acids to and absorption from the small intestine in beef steers (Lee et al., 2002). It has long been known that efficient microbial protein synthesis in the rumen requires a balanced supply of non-protein nitrogen (NPN; amino acids and ammonia) and energy (Beever, 1993). Unfortunately, this is not the case with traditional forages. Peptides and ammonia from soluble leaf proteins are rapidly released by the action of plant and microbial proteases (Kingston-Smith and Theodorou, 2000). However, the supply of rapidly available energy in the form of WSC tends to be limited and so the micro-organisms have to rely on structural carbohydrates in the cell (cellulose and hemicellulose) which are broken down at a much slower rate. Consequently this results in the loss of ammonia from the rumen and a reduction in efficiency of microbial protein synthesis. The increase in the supply of readily available energy (WSC) may improve this balance and consequently the efficiency of microbial protein synthesis, thereby reducing nitrogen loss from the rumen. This may explain the greater levels of performance from animals fed high WSC grass (MacRae et al., 1985). However, there is a degree of controversy as to whether the balance of energy and nitrogen release within the rumen can affect the efficiency of microbial protein synthesis. From their study with dairy cows, Herrera-Saldana et al. (1989) concluded, that a balance of readily degradable starch and protein fractions stimulated greater microbial protein flow than corresponding unbalanced diets. Other workers however, have reported only marginal or no effects in vivo (Henderson et al., 1998; Witt et al., 2000). In vitro, Newbold and Rust (1992) reported that an unbalanced versus a balanced supply of fixed amounts of protein and energy substrates had no lasting effects on bacterial growth. Their results agreed with the findings of Henning et al. (1991) who concluded that merely improving the degree of balance between rates of energy and nitrogen release in the rumen did not give predictable increases in microbial yield. This study, a two period RUSITEC (Czerkawski and Breckeridge, 1977) continuous culture experiment, was designed to investigate the effect of infusing additional levels of WSC, on top of a basal fresh perennial ryegrass (AberElan), on microbial parameters. The WSC infused into the RUSITEC apparatus was a mixture of disaccharide and polysaccharide sugars in a similar ratio to that found in Lolium perenne var. AberElan (80:20; fructan:disaccharides, mainly in the form of sucrose).

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2. Materials and methods 2.1. Experimental design The RUSITEC apparatus (Czerkawski and Breckeridge, 1977) was used to simulate a rumen environment in the laboratory. The experiment consisted of two 10 day periods with sampling of effluent for rumen parameters on days 9 and 10 of each period. The RUSITEC apparatus consisted of eight vessels and each period had four treatments, two vessels per treatment allocated at random. The treatments were a basal WSC concentration in the grass sample (Basal) and then the basal concentration plus a WSC infusate to raise the concentration of WSC by approximately 1.25×; 1.5× and 1.75×. 2.2. Experimental procedure The system consisted of eight airtight vessels immersed in a water bath maintained at 39 ◦ C. Two litres of rumen digesta were taken from two fistulated sheep, fed on standard grass silage, thoroughly mixed and transported to the laboratory (within 1 h) in a pre-heated vacuum flask. The rumen fluid was strained through a double layer of muslin into a CO2 -filled beaker, squeezing the muslin to obtain maximum liquid. Each vessel was charged with 500 ml of strained rumen liquor, 200 ml of artificial saliva (Durand et al., 1988), one nylon bag (pore size ca. 40 ␮m) containing ca. 80 g of rumen digesta solids (fibrous fraction from the rumen content straining) and another containing 80 g of fresh L. perenne var. AberElan, topped up with artificial saliva and sealed. Artificial saliva was pumped into the vessels at 0.5 ml/min by a peristaltic pump (202U, Watson-Marlow Ltd., Falmouth, Cornwall, UK) from 5 l stock solutions either alone or at three different concentrations of WSC. The control treatment was grass alone, i.e. with no extra WSC infused. The other three treatments were designed to deliver approximately 1.25×; 1.5× and 1.75× of the basal concentration of WSC in the grass, achieved by the infusion of extra WSC (Table 1). The basal sugar level determined from snip samples of the grass was 250 g/kg dry matter (DM), of which 80% was fructans and 20% di- and monosaccharide. The infusate was made up using sucrose as the disaccharide and, as grass fructans are difficult to extract in sufficient quantities, pure inulin, a fructan found in Jerusalem artichokes, was used as the fructan infusate. Inulin differs from fructan in its sugar moiety linkage and is a precursor for fructo-oligosaccharides. The results from a preliminary experiment showed an exponential disappearance of WSC from the grass samples. However, over the initial 14 h the decline was linear resulting in the loss of over 80% of the WSC. Therefore, for ease of Table 1 Daily input of WSC into the vessels (g per day) derived from the infusate and the basal grass Basal

Basal × 1.25

Basal × 1.5

Basal × 1.75

Basal grass WSC Infused inulin Infused sucrose

3.75 0 0

3.75 0.75 0.19

3.75 1.58 0.30

3.75 2.25 0.56

Total WSC

3.75

4.69

5.63

6.56

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design an infusion time of 14 h was conducted to simulate the disappearance of WSC from the experimental grass. This was followed by an infusion of artificial saliva (without added WSC) for the remaining 10 h, in each 24 h. The motor-driven arm moved the bags up and down through the rumen fluid/buffer mixture. As artificial saliva was infused the displaced effluent, and fermentation gases were collected in effluent collection vessels (cooled with ice) and gas collection bags, respectively. After 24 h the vessels were opened and the rumen digesta solids removed, squeezed and washed in artificial saliva. The washings were returned to the vessel and a new bag containing fresh grass inserted. On subsequent days the feed bag that had been in the vessel for 48 h was replaced with a bag containing fresh grass, as for the rumen digesta solids. 2.3. The grass The grass to be incubated in the culture vessels was cut daily (ca. 500 g) at 07:00 h and transported to the laboratory on ice. It was chopped using a Linhakker cutter before being weighed out into eight nylon bags (ca. 80 g per bag depending on the DM content, with a target DM input of 15 g per bag). A representative subsample was also taken for the determination of DM, total WSC, fibre fractions and total nitrogen concentrations. 2.4. The sampling period The experiment ran for two 10 day periods with sampling of liquid vessel contents for rumen parameters on days 9 and 10 at 0, 1, 2, 3, 4, 6, 14 and 24 h intervals from the addition of the new feed bag. The pH was measured using a Hydrus 400 pH probe (Fisher Scientific UK, Loughborough, Nottinghamshire, UK), 1 ml was taken and acidified with 100 ␮l of 2 M HCl and used for ammonia-N (NH3 N) analysis and another 1 ml was acidified with 100 ␮l of orthophosphoric acid and analysed for volatile fatty acid (VFA) concentration. At the end of each period the grass residues and supernatant from the vessels were collected for analysis. 2.5. Determination of microbial protein Ammonium 15 N sulphate ((15 NH4 )2 SO4 ) infused in the artificial saliva (Durand et al., 1988) was used as the microbial marker. At the end of the experiment the vessel supernatant and collected effluent were combined which contained the liquid associated bacteria (LAB). The solid associated bacteria (SAB) were harvested from the grass residues collected from the nylon bags on day 10 of the experiment. The grass residues were suspended in 100 ml of saline (9 g NaCl/l) in a Stomacher bag (Colworth 400, Colworth House, Bedford, Bedfordshire, UK), processed for 5 min to detach adherent bacteria. The liquid washings were retained and the residue processed again in the same manner, the combined washings providing a sample for harvesting SAB. The LAB and SAB samples were then combined to provide a total bacterial sample, which under-went two centrifugation regimes. Two hundred millilitres were centrifuged at 1500×g for 10 min and then the supernatant centrifuged at 30,000×g for 25 min followed by a distilled water wash and a further spin (30,000×g) to obtain a pure bacterial pellet. The total solids sample was produced in the same way without

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the initial 1500 × g spin. The pellets were then freeze-dried prior to analysis. These samples were analysed for total nitrogen and 15 N to determine microbial nitrogen enrichment using a mass spectrophotometer (ANCA/SL 20/20, PDZ Europa Ltd., Crewe, Cheshire, UK). Values for bacterial nitrogen per kg organic matter apparently digested were then calculated as described by Carro and Miller (1999). 2.6. Chemical and statistical analysis Water-soluble carbohydrate in the grass and residue was determined as described by Thomas (1977). Ash and by difference organic matter (OM) of the grass and residue was analysed by combusting at 550 ◦ C for 6 h in a muffle furnace. Volatile fatty acids (VFA) in the effluent liquor were determined by gas chromatography using Chrompack CP 9002 (CP-Sil 5CB column 10 m × 0.25 mm ID; Chrompack, UK) following the method of Zhu et al. (1996). In vitro dry matter digestibility (IVDMD) as described by Jones and Hayward (1975). Ammonia nitrogen was assessed enzymatically using glutamate dehydrogenase on a discrete analyser (FP-901 M Chemistry Analyzer, Labsystems Oy, Helsinki, Finland; Test kit No. 66–50, Sigma–Aldrich Co. Ltd., Poole, Dorset, UK). Total nitrogen was determined by a micro-Kjeldahl technique using ‘Kjeltec’ equipment (Perstorp Analytical Ltd., Maidenhead, Berkshire, UK). Neutral detergent fibre (NDF) was determined as described by Van Soest (1963) and acid detergent fibre (ADF) analysed according to the method of Van Soest and Wine (1967) using the Tecator Fibretec System equipment (Tecator Ltd., Thornbury, Bristol, UK). All measurements were blocked according to vessel and analysed using a general analysis of variance with polynomial contrast to compare water-soluble carbohydrate inclusion level (0, 1.25, 1.5, 1.75) × Period. For pH, ammonia and VFA of the vessels a daily mean was calculated and then ran through the model. The period effect and interaction were found not to be significant (P < 0.05) and so a mean of the two periods is presented (Genstat 5: Lawes Agricultural Trust, 1997).

3. Results The mean basal level of WSC in the grass was 250 (S.E. 10.0) g/kg DM with fibre contents of 202 (S.E. 7.5) and 411 (S.E. 13.2) g/kg DM for ADF and NDF, respectively. The nitrogen content of the grass was 21.0 (S.E. 0.47) g/kg DM with an in vitro dry matter digestibility of 67.9% (S.E. 0.66). The daily input of WSC into the vessels calculated from the basal sugar content of the grass (mean input of 15 g DM per day) and the amount of infused WSC per day are presented in Table 1. The changes in pH and ammonia-N and total VFA concentrations in effluent with time of incubation are shown in Fig. 1. The ammonia-N concentration and pH decreased with increasing WSC content for approximately 8 h. Thereafter there was a period of little change to 14 h followed by a gradual increase to complete the 24 h cycle. The corresponding mean values for VFA concentrations and pH of the effluent are presented in Table 2 along with daily effluent outflow rate from the vessels, the fraction liquid turnover rates of the vessels were not

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Fig. 1. Changes in rumen parameters in the effluent of in vitro continuous culture vessels over time on the four levels of WSC inclusion: Basal ( ); Basal × 1.25 ( ); Basal × 1.5 ( ) and Basal × 1.75 (×). (a) Ammonia-N concentration; (b) pH and (c) total VFA concentration (n = 8 ± S.E.M.).

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Fig. 1. (Continued ).

significantly different with a mean of 0.03 h. The pH values of effluent showed a significant linear reduction from Basal to Basal × 1.75 (P = 0.001). There was a trend (P < 0.1) for a quadratic effect for daily effluent flow increasing from Basal to Basal × 1.5 and thereafter declining. There were significant differences in the molar proportions of VFAs produced and Table 2 Daily flow rate, pH and VFA concentrations (mmol/l) in effluent from RUSITEC systems fed grass and infused with different levels of WSC Basal

Flow rate (l per day) pH Total VFA Acetate Propionate Butyrate Ratio P/(A + B) Molar proportions Acetate Propionate Butyrate

Basal × 1.25

Basal × 1.5

Basal × 1.75

S.E.D.

Significance T

L

Q

0.66 6.4

0.69 6.3

0.73 6.2

0.65 6.0

0.053 0.05

0.1 0.004

NS 0.001

0.1 NS

133.3 76.2 35.1 16.5 0.38

135.3 74.3 38.1 17.3 0.42

139.4 70.8 44.6 16.8 0.51

143.1 60.7 50.1 19.1 0.63

4.45 4.15 3.45 0.77 0.050

NS 0.015 0.015 0.024 0.016

0.04 0.05 0.004 0.023 0.003

NS NS NS NS NS

0.57 0.26 0.14

0.55 0.28 0.14

0.51 0.32 0.13

0.43 0.35 0.14

0.020 0.018 0.005

0.02 0.009 NS

0.005 0.002 NS

NS NS NS

T, L and Q: treatment, linear and quadratic effects, respectively, P/(A + B) = propionate/(acetate + butyrate). WSC levels are defined in Table 1.

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Table 3 Microbial N production, organic matter apparently digested, ammonia-N concentration and the efficiency of microbial protein synthesis in RUSITEC, fed grass and infused with different levels of WSC Basal

Basal × 1.25 Basal × 1.5 Basal × 1.75 S.E.D. Significance

Microbial N (mg per day) 144.6 143.0 OMAD (g per day) 14.4 13.2 Ammonia-N (mmol/l) 3.8 2.2 EMPS (g N/kg OMAD) 9.87 10.81

158.0 12.5 0.9 12.67

84.6 12.0 0.5 7.05

13.31 0.53 0.31 0.79

T

L

Q

0.001 0.004 0.001 0.001

0.002 0.001 0.001 0.014

0.003 NS 0.05 0.001

T, L and Q: treatment, linear and quadratic effects, respectively, OMAD: organic matter apparently digested, EMPS: efficiency of microbial protein synthesis. WSC levels are defined in Table 1.

total VFA production (P = 0.04). In particular the molar proportion of propionate increased (P = 0.02) and the molar proportion of acetate decreased (P = 0.009) with increasing levels of WSC inclusion. As a result of these changes the glucogenic:lipogenic VFA ratio (propionate/(acetate + butyrate), P/(A + B)) increased (P = 0.016) with addition of WSC. The daily flows of microbial nitrogen, organic matter apparently digested, ammonia-N and forage N incorporated into microbial protein per kg OM apparently digested/efficiency of microbial protein synthesis (EMPS) are presented in Table 3. For the first two increases in WSC level (Basal × 1.25 and Basal × 1.5) there was an increase in EMPS, but at the highest WSC addition there was a dramatic reduction in EMPS. This resulted in a significant linear (P = 0.014) and quadratic effect (P < 0.001). This pattern was repeated in the total daily microbial-N produced, again with significant linear (P = 0.002) and quadratic effects (P = 0.003). The OM apparently digested (grass OM + infused WSC) decreased stepwise from Basal to Basal × 1.75, despite the increase in OM input with increasing WSC inclusion, with a significant linear effect (P < 0.001). The ammonia-N concentration in the effluent also declined significantly with increasing WSC inclusion with significant linear (P = 0.001) and quadratic effects (P = 0.05). The chemical composition of the grass related to dry matter input after incubation in the RUSITEC system is presented in Table 4. With increasing WSC content there was a significant decline in the extent of fibre digestion (P = 0.012 and P = 0.019 for ADF and NDF, respectively). There was also an increase in WSC retained by the residues (P = 0.004), however no difference in total nitrogen content was observed. Table 4 Composition of grass residues in relation to dry matter input after incubation in RUSITEC for 48 h Basal

TN (g/kg DMI) ADF (g/kg DMI) NDF (g/kg DMI) WSC (g/kg DMI)

5.7 45.8 91.4 0.84

Basal × 1.25

5.8 58.9 104.1 1.18

Basal × 1.5

5.5 70.6 127.2 1.66

Basal × 1.75

5.7 79.9 143.3 1.79

S.E.D.

0.30 3.67 6.46 0.087

Significance T

L

Q

NS 0.012 0.019 0.004

NS 0.003 0.004 0.001

NS NS NS NS

T, L and Q: treatment, linear and quadratic effects, respectively. TN: total nitrogen; ADF and NDF: acid and neutral detergent fibre; WSC: water-soluble carbohydrate; DMI: dry matter input.

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4. Discussion The pH of the vessels declined for the first 8 h and then increased after 14 h of incubation, which corresponded with the end of the period of WSC infusion. The fact that the Basal treatment behaved in a similar manner to the other treatments indicated that constant infusion for 14 h closely simulated the rate of WSC release into the medium from the grass. The lowest pH was observed at the highest level of WSC inclusion. Henning et al. (1991), in an in vitro study, noted that at a “low” level of WSC inclusion pH did not decline below ca. 6.0, whilst at the “high” level there was a rapid decline to ca. 4.7. This decline in pH probably occurred as a result of the rapid fermentation of WSC, resulting in a rapid increase in VFA and lactic acid concentrations (Obara et al., 1991). Dawson and Allison (1988) suggested that a pH of around 5.5 represented the tolerance limit for many rumen micro-organisms and a pH of below 6.0 is less than optimal and would effect microbial synthesis. The reduction in pH, associated with increasing sugar inclusion from Basal to Basal × 1.75, was accompanied by a reduction in fibre digestion, which supports the findings of Grant and Mertens (1992), who showed an inhibition of cellulolytic, hemicellulolytic and pectinolytic organisms at low pH. Russell and Baldwin (1978) showed that for micro-organisms using WSC as an energy source, preference for certain substrates could cause shifts in the composition of the microbial population. Further shifts in the population would then be induced by inter-species competition (Russell et al., 1981). It was not possible to assess the extent of these changes in the current experiment, but evidence that fermentation shifts occurred are present in the changes in extent of digestion of the fibre fraction and changes in the molar proportions of VFA in the effluent. Despite the drop in pH with increasing WSC inclusion, the total VFA concentration increase between treatments was numerically small. This suggests that there was an increase in the production of lactate to account for the drop in pH. Some bacteria: Selenomonas ruminantium, Butyrivibrio fibrisolvens (Bauchop and Mountfort, 1981; Marvin-Sikkema et al., 1990) and holotrich protozoa: Dasytricha ruminantium, Isotricha spp. have been shown to produce lactate during carbohydrate fermentation. Despite these numerically small changes in total VFA concentration there were significant differences in the molar proportions and the production of the individual VFAs. There was a trend for the molar proportion of acetate to decrease and propionate to increase with increasing WSC inclusion. Obara et al. (1991) observed similar differences when infusing sucrose compared to a water supplement to the rumen of cattle. The patterns towards decreased acetate and increased propionate production reflect a gradual shift away from fibre digestion and towards fermentation of readily fermentable carbohydrates (WSC). The reduction in fibre digestion in response to WSC addition at Basal × 1.75 dramatically altered the VFA molar proportions, suggesting that changes in the microbial population had occurred, towards micro-organisms that had a greater reliance on WSC rather than fibre to supply energy (Russell and Baldwin, 1978). This may also explain the higher WSC content of the residues with greater sugar inclusion, as there may be less solid associated bacteria breaking down the cell walls to release the WSC from plant cells. An alternative hypothesis may be that increased WSC concentration of the medium could reduce WSC release from the plant cells by osmosis. However, the increase in propionate and reduction in acetate concentrations may have numerous benefits to the ruminant animal, in terms of increasing nitrogen and energy utilisation (MacRae and

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Lobley, 1986). In addition, Wolin et al. (1997) stoichiometrically determined that methanogeneis would be reduced when the ratio of acetate:propionate was reduced as occurred when increasing the WSC inclusion in this experiment. A significant decrease in ammonia nitrogen was observed in the vessels with increasing levels of WSC inclusion, with Basal × 1.75 averaging as low as 0.34 mmol/l. Satter and Slyter (1974) reported a range of 1.43–3.57 mmol/l required for optimum microbial protein synthesis. Therefore, the micro-organisms in the vessels on Basal × 1.5 and Basal × 1.75 may have been nitrogen deficient which would inhibit or limit microbial protein synthesis. However, some bacteria scavenge nitrogen as ammonia at concentrations as low as 0.1 mmol/l, such as Ruminococcus flavefaciens (Duncan et al., 1992). Henning et al. (1991) and Newbold and Rust (1992) also reported significantly lower ammonia concentrations on a higher energy diet under in vitro conditions. Henning et al. (1993) suggested that the lower ammonia-N concentration on the higher energy diet was a result of a greater microbial utilisation of dietary nitrogen from an improved energy and nitrogen synchronisation. Alternatively, at the higher level of WSC inclusion, less ammonia may be liberated as microbial and plant metabolic activity may have been affected by changes in the environment, e.g. pH (possibly through an increase in lactate production). This would appear to be the case in treatment Basal × 1.75 as the microbial nitrogen flow and EMPS were significantly lower. On the other three treatments there was a linear increase in EMPS with increasing WSC concentration (P = 0.014). Values were, however, lower than the reported recognised value of 30 g microbial nitrogen/kg organic matter apparently digested (OMAD; Agricultural Research Council, 1980), and may have been related to nitrogen limiting conditions, as ammonia-N concentrations were very low in the vessel over long periods during the day. The observed drop in efficiency at the highest level of sugar inclusion may also be related to this nitrogen deficiency in the vessels. It appears unlikely that the observed drop in EMPS was solely a result of the reduction in organic matter apparently digested as both the Basal × 1.5 and Basal × 1.75 treatments showed similar levels of OMAD. However, if there is insufficient nitrogen from the diet, which appears to be the case in this study in particular the Basal × 1.75, carbohydrate fermentation and microbial growth will become ‘uncoupled’ leading to a futile cycles of bacterial energy metabolism and a consequent reduction in EMPS. It may therefore be of interest to speculate a potential increase in EMPS if the vessels had provided extra degradable nitrogen. The lower EMPS may also have been the result of the lower flow rate from the highest WSC inclusion vessels, the reason for this reduced flow rate is unknown, but may have been related to the increased viscosity of the highest WSC infusion. A nitrogen limiting environment is less likely to occur in vivo as nitrogen would be recycled back into the rumen during such periods of shortage (Beever, 1993). This highlights the problems of extrapolating in vitro data to the in vivo situation. Further caution must be applied in relating this experiment to in vivo experiments on high WSC grass. Increasing the WSC supply may not only increase EMPS but may also cause a drop in cellulolytic activity, as reflected in the reduction in fibre digestion. Therefore, in vivo this may hamper not only the energy release from the gradual breakdown of hemicellulose and cellulose but also the further release of WSC from plant cells, which may reduce the EMPS. However, this reduction in cellulolytic activity will depend on how much the rumen environment is affected by the high WSC, pH, lactate and VFA concentration changes, which may not occur in the rumen due to greater homeostatic

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control. It is also important to note that an unusually high level of WSC was used in Basal × 1.75.

5. Conclusion This study has helped to determine the effects of increasing the concentration of WSC to the rumen microbial population under controlled conditions. An increase in the supply of WSC increases the glucogenic:lipogenic VFA ratio and reduces ammonia concentrations. In these respects the findings indicate potential for improved animal productivity on high WSC forages, through an increase in the efficiency of nitrogen and energy utilisation as a result of reduced rumen ammonia concentrations and the nature of the energy yielding VFAs absorbed from the rumen. The effect on the efficiency of microbial protein synthesis appears to be a result of a microbial population shift with increasing WSC inclusion.

Acknowledgements This work was funded by a LINK Sustainable Livestock Production programme involving the Ministry of Agricultural Fisheries and Food, Milk Development Council, Meat and Livestock Commission and Germinal Holdings Ltd. The authors are grateful to M.S. Dhanoa for his statistical advice, A.E. Brooks and D.K. Leemans for their assistance with the batch culture and RUSITEC procedures and J.K.S. Tweed and his staff for the analysis of the samples.

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