Effects of bacterial inoculation of unwilted and wilted grass silages. 1. Rumen microbial activity, silage nutrient degradability and digestibility

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1 Journal of Agricultural Science, Cambridge (1998), 131, Cambridge University Press Printed in the United Kingdom 103 Effects of bacterial inoculation of unwilted and wilted grass silages. 1. Rumen microbial activity, silage nutrient degradability and digestibility T. YAN *, D. C. PATTERSON,, F. J. GORDON, AND D. J. KILPATRICK The Agricultural Research Institute of Northern Ireland, Hillsborough, Co. Down, Northern Ireland BT26 6DR, UK The Department of Agriculture for Northern Ireland and The Queen s University of Belfast, Newforge Lane, Belfast BT9 5PX, UK (Revised MS received 20 January 1998) SUMMARY A set of four silages, comprising unwilted and wilted silages, both with and without bacterial inoculation, was prepared from perennial ryegrass swards on each of eight harvesting occasions during the 1994 growing season. The four silages, within a single harvest, were offered as the total diet at the maintenance level of feeding to 16 wether sheep in an 8-period change-over study with experimental periods of 3 weeks duration, in order to determine whole tract digestibility and urinary excretion of purine derivatives (PD). Silage dry matter (DM) and nitrogen (N) degradabilities were also examined in the rumen of four rumen-fistulated steers offered a medium quality silage and a concentrate supplement at a ratio of 60: 40 (silage: concentrate) on a DM basis. The data presented are the mean results obtained across the eight harvests. There were no significant effects of inoculation on silage ph and ammonia-n total-n across the unwilted and wilted materials. Inoculation of either the unwilted or wilted silages had no significant effect on microbial activity in the rumen of sheep, as indicated from excretion of urinary PD, or on silage DM or N degradability assessed with rumen-fistulated steers. However, inoculation significantly increased the digestibility of organic matter (1 7%, P 0 01), N (4 6%, P 0 001), energy (2 3%, P 0 01), neutral detergent fibre (1 5%, P 0 05) and acid detergent fibre (2 8%, P 0 001) in the silage. These increases in digestibility following inoculation, in general derived equally from both the unwilted and wilted silages. The results indicate that the increase in silage nutrient digestibility following inoculation probably reflects a more extensive digestion in the abomasum and intestine, rather than in the rumen. Wilting of grass prior to ensiling resulted in silages with significantly lower ph (5 4%, P 0 05) and ammonia-n total-n (42 7%, P 0 001) across the untreated and inoculant-treated materials. Wilting also significantly increased (P 0 05) urinary PD-N output of sheep by 6 9% and silage DM degradability by steers by % across the untreated and inoculant-treated silages. However, there were no significant effects of wilting on silage N degradability and silage nutrient digestibility. INTRODUCTION Bacterial inoculation of unwilted grass silage usually results in more rapid production of lactic acid and more rapid decline in ph in the silage during the first few days post-ensiling (Gordon 1989; Mayne 1990; Yan et al. 1996). This would minimize proteolysis and the loss of available nutrients in the silage. An examination of 25 comparisons published since 1989 * To whom all correspondence should be addressed. tyan alpha1.dani.gov.uk has shown that inoculation increased the concentration of water-soluble carbohydrates (WSC) by proportionately (S.D ) and decreased ammonia-n total-n by (S.D ) in grass silages (Anderson et al. 1989; Gordon 1989; Steen et al. 1989; Mayne 1990, 1993; Martinsson 1991; Chamberlain et al. 1992; Smith et al. 1993; Keady & Steen 1994, 1995; Keady et al. 1994; Sharp et al. 1994; Keady & Murphy 1996, 1997; Yan et al. 1996). Such effects of inoculation may therefore lead to a greater microbial activity in the rumen by provision of more fermentable metabolizable energy and more

2 104 T. YAN ET AL. preformed amino acids. Sharp et al. (1994) reported a trend indicating an increased efficiency of microbial protein synthesis within the rumen of heifers offered inoculant-treated silages, using incorporation of S for the measurement of microbial protein in digesta. An increase in digestible organic matter in dry matter (D-value) following inoculation, as noted in a review by Mayne & Steen (1993), and also reported by Yan et al. (1996), may indicate a greater microbial activity in the rumen of animals offered inoculant-treated silages. However, there has been no information available in the literature on the effect of inoculation on rumen microbial activity as indicated by urinary excretion of purine derivatives. As wilting of grass prior to ensiling increases grass WSC concentration, inoculation would be more likely to achieve a better fermentation pattern in the silage when used with the wilted material (Done 1986). A small number of studies have indicated decreases in ph and ammonia-n total-n in silages following inoculation of wilted grass (Martinsson 1991; O Kiely 1994; Yan et al. 1996). Inoculation of wilted forages has been reported to increase silage nutrient digestibility with sheep (Martinsson 1991) and in an in vitro study (Chen et al. 1994). However, Yan et al. (1996) observed no significant difference in mean nutrient digestibility with sheep across three comparisons of inoculated against untreated grass silages. The present study was therefore designed to evaluate the mean response to four commercial inoculant additives, based on selected strains of homofermentative bacteria, when used with unwilted and wilted grasses. The current paper presents data on the effects on silage fermentation, urinary excretion of purine derivatives, nutrient digestibility and degradability. The effects on silage DM intake, milk production and eating behaviour of dairy cows will be reported in a subsequent paper (Patterson et al. 1998). In order to provide a broadly based view of the effects, the comparisons were carried out on eight separate harvesting occasions, i.e. one inoculant used with two harvests, over a single silage-making season. MATERIALS AND METHODS Silage preparation A set of four silages was prepared on each of eight harvesting occasions within the 1994 silage-making season. The four silages comprised unwilted and wilted silages, both with and without application of one of four lactic acid producing bacterial inoculants at ensiling. Each inoculant was thus used with two harvests, giving an overall total of 32 (4 8) silages made over the eight harvests. The eight harvests were spread over three primary growths, three first regrowths and two second regrowths of perennial ryegrass. On each occasion the total experiment area of grass was mown with the same mower fitted with a V-spoke grass conditioner (Taarup Model 307). Within 30 min of mowing, half of the area was lifted using two similar precision-chop forage harvesters (Reco-Mengele models SH30N and SH40N) which were adjusted to produce the same chop length and equipped for additive application. The other half of the crop was immediately spread to cover the total ground area from which it was cut in order to maximize drying rates. On each occasion the grass for wilting was retedded next morning and lifted when it achieved a DM concentration of c. 320 g kg (range g DM kg) by the same two forage harvesters as used with the unwilted crop. No rainfall occurred during wilting at any of the eight harvests and wilting required 30, 72, 45, 28, 27, 30, 30 and 49 h for harvests 1 8 respectively, with a mean wilting period of 39 h (S.D. 15 7). The two harvesters picked up alternate rows of grass during each harvesting operation. One harvested untreated grass (control), while the other harvested material treated with one of the four inoculants (additives). The four inoculants contained different ingredients, but each had Lactobacillus plantarum. Prior to harvesting, the inoculants were reconstituted in water according to the manufacturer s recommendations, and then applied via a metering pump which discharged into the auger chamber of the harvester. At each harvest, c. 40 t of herbage from each of the two unwilted treatments and 20 t from each of the two wilted treatments were ensiled in roofed, concrete lined, trench silos and covered with plastic sheeting, which was weighted by a complete covering of tyres. Ensiling, compaction and sealing were completed in each instance within an 8 h period. The weight of each load of grass was recorded and a representative sample (1 kg) was taken from each load prior to ensiling. Each sample was analysed for buffering capacity and concentrations of DM, nitrogen (N), acid detergent fibre (ADF), neutral detergent fibre (NDF), ash, water-soluble carbohydrates (WSC) and gross energy (GE). All procedures for chemical analysis of the grass were as described by Mayne & Gordon (1984). Sampling of silages and silage aerobic stability assessment Prior to the start of each period in the main feeding experiment with dairy cows, the silage was completely removed from each of the relevant silos and was completely mixed in a forage mixer wagon. A series of wooden boxes (400 kg capacity) were prepared with double polythene liners of 110 µm thickness. The silage from each silo was then discharged into 46 boxes by means of eight fillings, which gave a sandwich of eight shallow layers of the silage in each box. Immediately following the completion of filling the boxes, each of the two liners in each box was

3 Inoculation of unwilted and wilted grass evacuated and sealed. Two boxes of each silage were randomly chosen and the silage in these two boxes was completely mixed together for sampling for chemical analysis, aerobic stability assessment and rumen degradability study. The remaining silage from these two boxes was then used in the studies of total tract digestibility and urinary excretion of purine derivatives with sheep. Three representative samples of fresh silage were taken for determination of oven DM, and the dried samples were then used for determination of ash, ADF and NDF. A further three samples of fresh silage were also taken for measurements of toluene DM, ph, total N, ammonia N, lactate, acetate, propionate, butyrate, valerate, ethanol, propanol and WSC. The methods adopted for the above analysis were as described by Mayne & Gordon (1984). The fresh samples were also used for determination of acid insoluble N (AIN) using the method described by Brady (1960). A total of 6 6 kg samples of each silage were assessed for aerobic stability. Three samples of each silage were each placed loosely in a polythene lined polystyrene box and each sample was tossed ( fluffed up ) once daily in the morning for measurement of both ph and temperature change in the silage (fluffed silages), using the techniques described by Mayne (1993). The remaining three samples were assessed for temperature change only using the procedure described above, except that the silage was fluffed just once at the time of filling of each box to equalize the distribution of the silage within the box. The silage was then compressed by placing a ballast of 20 kg on the top of a rigid plywood former to consolidate the silage for 24 h, after which both the ballast and the plywood former were removed (compressed silages). The silage remained in situ in an unfluffed state for the remainder of the measurement period. Silage nutrient digestibility and urinary excretion of purine derivatives by sheep The 32 silages, which were made from the eight harvests, were offered alone to 16 castrated male sheep (1 year old) in a change-over design experiment using 8 periods (i.e. one harvest per period), each of 3 weeks duration, giving four animals per treatment in each period. The four silages within each harvest (2 additive 2 wilting treatments) were evaluated within a single period. Each of the four silage samples had been obtained by choosing two boxes of the silage at random during the box filling operation for the main feeding trial as described in the previous section. After thorough mixing of the silage, amounts sufficient to meet the daily maintenance allowance of each animal were weighed into polythene bags and stored at 20 C until 24 h prior to feeding. The silages were offered to the sheep once daily in the morning without any supplement. The sheep were confined individually in pens during the first 2 weeks and in metabolism crates during the third week. The crates were designed for separation of faeces and urine. Total faeces and urine were collected during the final 6 days of each 3-week period. Faeces were weighed daily, stored at 2 4 C and bulked for each animal at the end of the 6-day collection period. The fresh faeces were then completely mixed and divided into two portions. One portion was analysed for fresh N concentration and the remaining portion was dried at 100 C over 48 h for determination of DM concentration. The dried sample was milled through a 1 mm screen and analysed for GE, ADF, NDF and ash. Urine output was recorded twice daily, namely, in the morning and afternoon, and a sample of 5% (w w) from each collection was frozen at 20 C without preservatives. The urinary concentrations of allantoin, uric acid, xanthine and hypoxanthine (purine derivatives, PD) were determined by using reverse-phase high performance liquid chromatography (reverse-phase C18 column supplied by Shandon, Runcorn, Cheshire, UK). PD detection was via a UV spectrophotometer (LDC Spectromonitor III supplied by LDC, Milton Keynes, UK) at a wavelength of 218 nm. Microbial N synthesis in the rumen of sheep was estimated using the factors proposed by Chen et al. (1991). Fermentable metabolizable energy (FME) concentration in the silage was estimated using the methods of the AFRC (1992), and effective rumen degraded dietary N (ERDN) concentration in the silage was calculated using the equation of the AFRC (1992) and silage N degradation factors obtained in the present study. Silage DM and N degradability by cattle The 32 silages, which were made from four inoculants, each used with two harvests, each harvest comprising four silages (unwilted and wilted grass both with and without inoculation), were also examined with four rumen-fistulated steers in a 4-period study. During each period, three bags of each of eight silages, which were made within one inoculant as four silages (within one harvest) two harvests, were suspended in the rumen of each animal for 0, 4, 8, 12, 24, 48 and 72 h, respectively. Twenty-four bags (3 bags 8 silages) were thus placed in the rumen of each of the four animals in the morning before feeding at each incubation time. At the end of the arranged incubation time as indicated previously, all the 24 bags were removed and a further set of 24 bags was then placed in the rumen of each animal next morning before feeding, for a subsequent incubation time. The animals were offered a non-experimental silage of medium

4 106 T. YAN ET AL. quality ad libitum and a concentrate supplement on the basis of a silage:concentrate ratio of 60:40 (DM basis) for 3 weeks prior to start of the incubation of silages and then throughout this study. The techniques adopted for determination of silage DM and N degradability were as described by Ørskov & McDonald (1979). Statistical analysis The data on grass composition were analysed as a randomized block design with harvest dates as blocks. The data on silage composition and silage aerobic stability assessments were analysed each as a 2 (inoculant) 2 (wilting) randomized block factorial design experiment, with harvest dates as blocks. The data on urinary excretion of purine derivatives and digestibility were analysed using a two-stage procedure. First the 128 individual observations (16 sheep 8 periods (harvests)) were analysed to estimate means for the 32 silages adjusted for animal effects. These 32 adjusted means were then analysed by analysis of variance using a randomized block model, with harvest dates as blocks and treatments in a 2 inoculant 2 wilting factorial arrangement. This model assumes no harvest treatment interaction and provides a test of inoculation and wilting effects over a range of different harvest dates. The same procedure was also used to analyse the data obtained in the silage DM and N degradability study with rumen-fistulated steers. RESULTS A total of 32 silages, prepared from eight harvests, was examined and all the results reported are mean values across the eight harvests. Grass composition Wilting significantly increased the concentration of Table 1. Mean data for grass composition (g kg DM, unless otherwise stated) Unwilted Wilted S.E. (D.F. 7) DM (g kg) Ash Crude protein ADF NDF GE (MJ kg DM) WSC Buffering capacity (meq kg DM) DM in grass at ensiling by 186 7% (P 0 001), but had no significant effect on mean concentration of ash, CP, ADF, NDF, GE, WSC or buffering capacity in grass (Table 1). Silage aerobic stability assessment There were significant interactions between inoculation and wilting for days to ph or temperature rise, days to temperature maximum and cumulative temperature rise to day 5 (P 0 01) in the fluffed silages (Table 2). With the fluffed silages, in terms of main effects across the unwilted and wilted silages, inoculation significantly decreased days to temperature rise by 11 1% (P 0 05) and days to temperature maximum by 11 5% (P 0 05), but increased cumulative ph or temperature rise to day 5 by 39 6% (P 0 05). However, by examining individual treatment effects, these effects mainly arose from the wilted silages. With the fluffed silages, in terms of main effects across the untreated and inoculant-treated silages, wilting significantly increased (P 0 05 or less) days to ph (22 6%) or temperature rise (23 5%), days to ph (35 3%) or temperature maximum (34 0%) and maximum ph rise (1 7%), while it decreased maximum temperature rise (4 7%) and cumulative ph (32 7%) or temperature (20 9%) rise to day 5. These effects in general mainly derived from the untreated silages. The effects of inoculation and wilting on the temperature changes in the compressed silages were similar to the fluffed silages. Silage composition A significant interaction was obtained between inoculation and wilting on mean NDF concentration (P 0 05) in the silages (Table 3). In terms of main effects across the unwilted and wilted silages, inoculation had no significant effect on any variable investigated, except for acetic acid concentration which was significantly reduced by 25 3% following inoculation (P 0 05). However, in terms of main effects across the untreated and inoculant-treated silages, wilting significantly influenced (P 0 05 or less) all variables investigated except for lactic acid concentration. The variables which were significantly higher (P 0 05 or less) following wilting were the concentrations of DM (79 6%), ash (12 0%), CP (2 4%), WSC (302 0%) and AIN (10 4%). Otherwise those variables which were significantly lower (P 0 05 or less) following wilting were ph (5 4%), ammonia-n total-n (42 7%), buffering capacity (39 4%) and concentrations of GE (3 2%), ADF (15 0%), NDF (8 1%), acetic acid (62 8%), propionic acid (86 0%), butyric acid (78 8%), ethanol (24 4%) and propanol (94 1%).

5 Inoculation of unwilted and wilted grass Table 2. Mean results of aerobic stability assessment Unwilted Wilted S.E. (W A)* Control Additive Control Additive (D.F. 21) ph fluffed silages Days to ph rise Days to ph maximum Maximum ph rise Cumulative ph rise to day Temperature fluffed silages Days to temperature rise Days to temperature maximum Maximum temperature rise ( C) Cumulative temperature rise to day 5 ( C) Temperature compressed silages Days to temperature rise Days to temperature maximum Maximum temperature rise ( C) Cumulative temperature rise to day 5 ( C) * Wilting Additive. Table 3. The mean composition of silages as removed from silos (g kg DM, unless otherwise stated) Unwilted Wilted S.E. (W A)* Control Additive Control Additive (D.F. 21) Alcohol corrected toluene DM (g kg) ph NH -N (g kg total N) Crude protein Gross energy (MJ kg DM) Acid detergent fibre Neutral detergent fibre Water soluble carbohydrates Acid insoluble nitrogen Ash Buffering capacity (meq kg DM) Lactic acid Acetic acid Propionic acid Butyric acid Ethanol Propanol * Wilting Additive. Silage nutrient digestibility through sheep There was no significant interaction between inoculation and wilting for any variable investigated (Table 4). In terms of main effects across the unwilted and wilted silages, inoculation had no significant effect on DM digestibility or D-value in the silages, while it significantly increased digestibilities of other nutrients investigated (P 0 05 or less). Within the unwilted silages, inoculation significantly increased digestibilities of DM (2 5%, P 0 05), OM (2 2%, P 0 05), N (5 7%, P 0 01), GE (2 8%, P 0 01), NDF (1 8%, P 0 05) and ADF (3 3%, P 0 001) in the silages. Similarly, within the wilted silages, inoculation significantly increased digestibilities of N by 3 4% (P 0 05) and ADF by 2 4% (P 0 001) in the silages. In terms of main effects across the untreated and inoculant-treated silages, wilting had

6 108 T. YAN ET AL. Table 4. Effects of wilting and inoculation on silage nutrient digestibility Unwilted Wilted S.E. (W A)* Control Additive Control Additive (D.F. 21) Dry matter Organic matter Nitrogen Gross energy Neutral detergent fibre Acid detergent fibre D-value * Wilting Additive. Table 5. Effects of wilting and inoculation on excretion of purine derivatives in urine of sheep* Unwilted Wilted S.E. (W A) Control Additive Control Additive (D.F. 21) Allantoin (mmol day) Uric acid (mmol day) Hypoxanthine xanthine (mmol day) PD-N (g day) Microbial-N (g day) Microbial-N kg Microbial-N ERDN (g g) Microbial-N FME (g MJ) Microbial-N DOM (g kg) *kg, metabolic liveweight; ERDN, intake of effective rumen degraded dietary N; FME, intake of fermentable metabolizable energy; DOM, digestible OM. Wilting Additive. no significant effect on any variable investigated, although wilting within the untreated silages significantly increased silage DM digestibility by 2 5% (P 0 05). Excretion of purine derivatives in urine of sheep There was no significant interaction between inoculation and wilting for any variable investigated (Table 5). Inoculation of either the unwilted or wilted silages had no significant effect on any variable investigated. However, in terms of main effects across the untreated and inoculant-treated silages, wilting significantly increased urinary PD-N output by 6 9% (P 0 05) and estimated microbial-n synthesis by 6 4% (P 0 05), and decreased microbial-n FME by 10 0% (P 0 01). These effects for the former two variables were only significant within the inoculanttreated silages (P 0 05), while for the latter variable the significant effect occurred within the untreated silages (P 0 05). DM and N degradability in the rumen of steers There was no significant interaction between inoculation and wilting on any variable investigated for either DM or N degradation in the silages (Table 6). Inoculation of either the unwilted or wilted silages had no significant effect on either DM or N degradability in the silages. Wilting with either the untreated or inoculant-treated silages also had no significant effect on silage N degradability. However, in terms of main effects across the untreated and inoculant-treated silages, wilting significantly (P 0 05) increased the readily soluble proportion of DM (a-value) by 3 0%, the fractional degradation rate of the potentially degradable DM (c-value) by 3 5% and DM degradability in the silages by 1 6, 2 2 and 2 4% respectively, when assuming fractional outflow rates from the rumen of 0 02, 0 05 and 0 08 h (AFRC 1992), respectively. These increases mainly arose within the untreated silages (P 0 05) because within the inoculant-treated silages wilting had no significant effects on silage DM degradability.

7 Inoculation of unwilted and wilted grass Table 6. Effects of wilting and inoculation on silage DM and nitrogen degradability in the rumen of steers Unwilted Wilted S.E. (W A)* Control Additive Control Additive (D.F. 21) Dry matter a-value b-value c-value Degradability r r r Nitrogen a-value b-value c-value Degradability r r r * Wilting Additive. r,r and r, the fractional outflow rates from the rumen of 0 02, 0 05 and 0 08 h, respectively (AFRC 1992). DISCUSSION The results obtained in the present study have indicated relatively similar effects of inoculation with unwilted and wilted silages, and there was no significant interaction between inoculation and wilting on any variable investigated for rumen microbial activity and silage nutrient degradation and digestion. Effect of inoculation The bacterial inoculation of the unwilted and wilted grass in the present study in general had little effect on mean silage fermentation characteristics across the eight harvests. The ph and ammonia-n total-n were similar following inoculation, although the lactic acid concentration tended to be increased. These effects within the wilted grass are relatively similar to those obtained previously (Martinsson 1991; Jones et al. 1992; O Kiely 1994; Chen et al. 1994; Yan et al. 1996). However, the responses to inoculation of the unwilted grass differ from those obtained in previous studies, which in general have indicated decreases in both ph and ammonia-n total-n in the silages following inoculation, both at this Institute (Gordon 1989; Mayne 1990; Yan et al. 1996) and elsewhere (Martinsson 1991; Smith et al. 1993; Sharp et al. 1994). The different results with the unwilted grass obtained in the present study could reflect low mean concentrations of DM (148 g kg) and WSC (21 g kg on a fresh basis) in the unwilted grass across the eight harvests, which would generally classify these materials as likely to produce poor fermentation patterns. Under such circumstances Henderson & McDonald (1984) and Done (1986), from reviews of previous information, suggested that the application of inoculants to such grasses at ensiling may not improve silage quality due to the limitation of fermentable substrate. Although inoculation of the unwilted and wilted grass had little effect on silage fermentation in the present study, silage nutrient digestibilities of OM, energy, N, ADF and NDF were significantly increased following inoculation across the eight harvests. These increases following inoculation are relatively similar with the unwilted and wilted silages and concur with those obtained in some previous studies with unwilted grass (Gordon 1989; Mayne 1990; Smith et al. 1993; Yan et al. 1996) and with wilted grass (Martinsson 1991; Chen et al. 1994). However, other previous studies with unwilted grass have indicated no effects of inoculation on silage nutrient digestibility (Rooke et al. 1988; Steen et al. 1989) or even reduced digestibility (Mayne 1993). In a similarly designed experiment to the present study, Yan et al. (1996) reported a trend indicating decreased silage OM, N and energy digestibilities following inoculation of wilted silage across three harvests. These effects disagree with those obtained in the present study. The reason for the differing effects of inoculation on the nutrient digestibility of wilted silage could derive from the different techniques used for removing the silages from opened silos in the two studies. In the present study all procedures for silo opening, silage removal, silage bagging and storing in a deep freeze environment were completed

8 110 T. YAN ET AL. within 7 h, while in the previous study (Yan et al. 1996) silages for digestibility were only removed from opened silos at intervals during the feeding out period. Therefore, in the previous study (Yan et al. 1996) the inoculant-treated silages may conceivably have had more aerobic deterioration than the untreated silages, since inoculation resulted in fewer days to rises in ph and temperature, and greater cumulative rises in ph and temperature to day 5 during the aerobic stability assessment in both the present study and the previous study and the previous experiment (Yan et al. 1996). The greater rates of aerobic deterioration with the inoculant-treated silages are likely to be due to the fact that some lactic acid bacteria can, under conditions of hexose limitation, metabolize lactic acid as an energy source under aerobic conditions (Lindgren et al. 1990). This produces acetate and formate and brings about an increase in ph and permits undesirable microorganisms to become active. On the other hand, in both the present and previous studies (Yan et al. 1996), the higher WSC and lactic acid concentrations of the inoculant-treated wilted silage may have served as energy sources for the growth of yeasts and moulds (Thomas & Morrison 1982) and led to greater rates of aerobic deterioration. In the present study the estimated microbial protein synthesis in the rumen of sheep was unchanged following inoculation of the unwilted or wilted grass across the eight harvests, as estimated from excretion of purine derivatives (PD) in the urine of sheep (Chen et al. 1991). Even within any individual harvest, PD output did not differ significantly between the inoculant-treated and untreated silages. The silage DM and N degradabilities were therefore similar following inoculation of the unwilted or wilted grass across the eight harvests, when assuming the rumen outflow rate of either 0 02 or 0 05 or 0 08 h (AFRC 1992). While Keady & Steen (1994), and Keady et al. (1994) also observed no significant change in silage DM degradability following inoculation of unwilted grass, silage DM digestibility over the whole tract was significantly higher with inoculant-treated than untreated silages, when the silages were offered to sheep as sole diets. The above results indicate that the increase in silage nutrient digestibility following inoculation of the unwilted and wilted grass probably reflects a more extensive digestion in the abomasum and intestine, rather than in the rumen. Effect of wilting Wilkins (1984) reviewed a large number of experiments and concluded that wilting increased silage ph from 4 4 to4 6 and decreased ammonia-n total-n from to A recent study carried out by O Kiely (1994) reported that wilting increased both ph and ammonia-n total-n with grass silages containing 208, 368 and 511 g DM kg. However, in the present study, wilting significantly decreased both ph and ammonia-n total-n across the untreated and inoculant-treated silages over the eight harvests. The reason for the reduction in ph is unclear, but may reflect the fact that the mean DM concentrations of 176 and 316 g kg in the unwilted and wilted silages, respectively, in the present study were both lower than those (212 and 350 g kg respectively) in the review of Wilkins (1984), but may also suggest that an improved wilting system was adopted in the present study. This system, which includes conditioning of the grass during cutting, immediate spreading and repeated tedding of the crops during the wilting period (Patterson 1993), has been shown to achieve faster rates of wilting than conventional systems used at this Institute (Yan et al. 1996). By using this improved wilting system, Yan et al. (1996) also observed a similarly low ph (3 95) in the wilted v. unwilted silages across the untreated and inoculant-treated silages over three harvests. In the present study, wilting resulted in silages with increased concentrations of WSC of 23 g kg DM and non-ammonia N of 2 1 g kg DM over the unwilted silages. Such effects could provide more FME and more preformed amino acids for the animals offered the wilted silages than those offered the unwilted silages. This would stimulate microbial activity in the rumen of animals offered the former silages, provided that the ERDN intake is sufficient, since some rumen microbial species have specific requirements for certain amino acids, such as methionine (Demeyer 1981). In the present study, microbial activity was estimated to be greater (P 0 05) in the rumen of sheep offered the wilted rather than the unwilted silages, using urinary PD excretion as an indicator of microbial growth. This increase was quantified as 0 63 g day of estimated microbial N in sheep. Therefore, rumen DM degradability in the silage was significantly increased following wilting when assessed with rumen-fistulated steers. When examining the individual treatment effects in the present study, the increase in silage DM degradability following wilting derived mainly from the untreated silages, since within inoculant-treated silages the increase in this variable following wilting was not significant. A similar result was also found with the silage DM digestibility. These differing effects of wilting with and without inoculation at ensiling may partly reflect the differences in improvement of silage aerobic stability by wilting. Wilting without inoculation significantly reduced the rates of aerobic deterioration of the silages, e.g. more days to ph or temperature rise and less cumulative ph or temperature rise to day 5, while the improvements were not significant following wilting when the inoculant additives were used at ensiling. A similar effect of wilting on silage DM digestibility across three harvests

9 Inoculation of unwilted and wilted grass was also reported by Yan et al. (1996) in a previous study with a similar design to the present study. Wilkins (1984) reviewed a number of earlier studies involving a range of silage additives at ensiling and reported a proportionate reduction of in silage DM digestibility of wilted silage v. unwilted silage as assessed with sheep. Rohr & Thomas (1984) also reported an average decrease of in silage DM digestibility due to wilting as assessed with sheep, in a review of the Eurowilt series of experiments. However, in the present study, no adverse effects of wilting on the silage DM digestibility (even higher when wilting without inoculation) probably reflect that an improved wilting system (Patterson 1993) was adopted in the present study as discussed previously. This system has resulted in considerably faster drying rates than those recorded in the early wilting studies at this Institute (Patterson et al. 1996). Therefore, in the present study, wilting resulted in silages with significantly lower ph and ammonia-n total-n, and significantly higher protein and WSC concentrations. The authors wish to acknowledge the provision of financial support for this study from J. Bibby Agriculture Ltd, Peterborough; Britmilk, Collin, Dumfriesshire; Christian Hansen (UK) Ltd, Reading; Microbial Developments Ltd, Malvern; Pioneer Hi- Bred (UK) Ltd, Hartford; Richardsons Fertilizers, Belfast; Zeneca Bio Products, Billingham. The authors also wish to thank the staff of the Agricultural Research Institute of Northern Ireland for their conscientious support throughout this study. AGRICULTURAL AND FOOD RESEARCH COUNCIL (1992). Technical Committee on Responses to Nutrients. Report No. 9. Nutritive Requirements of Ruminant Animals: Protein. Nutrition Abstracts and Reviews, Series B 62, ANDERSON, R., GRACEY, H. I., KENNEDY, S. J., UNSWORTH, E. F. & STEEN, R. W. J. (1989). 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