The effect of method of forage preservation on
the protein degradability and microbial
protein synthesis in the rumen
J. VerbicÏ
a,*, E.R. érskov
b, J. ZÏgajnar
c, X.B. Chen
b,
Vida ZÏnidarsÏicÏ-Pongrac
aaAgricultural Institute of Slovenia, 1000 Ljubljana, Hacquetova 17, Slovenia bRowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK cUniversity of Ljubljana, Biotechnical Faculty, Zootechnical Department,
1320 DomzÏale, Groblje 3, Slovenia
Received 15 May 1998; received in revised form 2 March 1999; accepted 3 September 1999
Abstract
Direct cut silage (DC), formic acid treated silage (FA), wilted silage (W), highly wilted silage (HW) and hay (H) were prepared from the same parental grass. In one experiment, the five diets were fed to sheep and the efficiency of rumen microbial protein synthesis and rumen digesta passage rate were determined. In a second experiment, organic matter and protein degradability characteristics in the rumen of sheep were determined by the in sacco method. Microbial protein supply (MN) was significantly higher (P< 0.05) in HW and H (12.63 and 12.77 g N kgÿ1 DM intake) than in DC, FA and W (11.09, 11.32 and 10.89 g N kgÿ1 DM intake). MN tended to be
negatively correlated to the concentration of total acids in the feeds (r ÿ0.73,P> 0.1). There were no significant differences in the digesta passage rates. Effective protein degradability (EDGCP)
were 855, 800, 796, 750 and 677 g kgÿ1for DC, FA, W, HW and H, respectively. Effective organic matter degradability (EDGOM) for DC, FA, W, HW and H were 552, 517, 526, 484 and 522 g kgÿ1,
respectively. A synchrony index (IS), which takes into account the rate of degradation of nitrogen
and organic matter, was calculated. The index varied significantly among the different preservation treatments (0.22, 0.34, 0.28, 0.35 and 0.87 in DC, FA, W, HW and H, respectively). MN tended to be positively correlated toIS(r0.72,P> 0.1). Estimated metabolizable protein concentrations
were 53.4, 59.9, 57.1, 70.3 and 75.3 g kgÿ1DM for DC, FA, W, HW and H, respectively. It was
concluded that, based on the protein value, hay was better than silages and wilted silages better than
82 (1999) 195±212
*Corresponding author. Tel.:386-61-1375375; fax:386-61-1375413
E-mail address: joze.verbic@kis-h2.si (J. VerbicÏ)
unwilted silages ensiled without an additive. The protein value of silages can be increased by the formic acid treatment prior to ensiling.#1999 Elsevier Science B.V. All rights reserved.
Keywords: Grass silage; Hay; Protein; Degradation; Microbial synthesis
1. Introduction
In the Central European climate, forage is usually preserved for at least 6±7 months of the winter period. It has been known for a long time that forage undergoes considerable changes during preservation. Either the forage is preserved as hay or, as silage, at least part of the protein is broken down, and some sugars are lost due to the action of plant enzymes in the field after harvest. When ensiled, additional changes occur during fermentation in the silo. Sugars are mostly fermented into organic acids and free amino acids are broken down to ammonia and some other non-protein compounds. Due to a relatively high crude protein concentration, it is expected that forage from grassland will be sufficient to meet the protein requirements for ruminants. However, from the results of Gill et al. (1987) and Dawson et al. (1988), it is evident that in some cases the protein from grass silage does not fulfil the protein requirements of growing cattle nor does it allow maximal microbial growth in the rumen. From the literature, it appears that the problem of inefficient protein utilization is more serious in silages than in hay or green fodder. In comparison with green fodder or hay, silage protein is more extensively degraded in the rumen (Merchen and Satter, 1983; Siddons et al., 1990; van Vuuren et al., 1990; LoÂpez et al., 1991; Jaakkola and Huhtanen, 1993; AufreÁre et al., 1994). Based on a limited number of observations, ARC (1984) suggested that the efficiency of microbial protein synthesis in fermented forages was about 30% lower than for fresh or dry forages. Ruminant protein systems currently used (PDI, VeÂrite and Peyraud, 1988; Metabolisable Protein System, AFRC, 1992; DVE, Tamminga et al., 1994) take into account that products of ensiling fermentation do not contribute to fermentable energy for microbial protein synthesis in the rumen. However, information on the effect of preservation methods on the microbial protein synthesis is still limited. In published experiments, the effect of preservation methods is often confounded by the effect of dry matter intake (Narasimhalu et al., 1989; Teller et al., 1992) and possible differences are often hidden by the supplementation with concentrates (Merchen and Satter, 1983; Teller et al., 1992; Jaakkola and Huhtanen, 1993; Jaakkola et al., 1993).
The objective of the present work was to compare five different preservation methods commonly used on farms (direct cut silage, formic acid treated silage, wilted silage, highly wilted silage and hay), based on the protein and organic matter degradation characteristics and efficiency of microbial protein synthesis in the rumen. The work was done in a climate that offered relatively good wilting conditions.
2. Material and methods
Two experiments were conducted. In Experiment 1, the feeds from the five different preservation methods were fed to four sheep. The efficiency of microbial protein
synthesis and rumen digesta passage rates were determined. In Experiment 2, the rumen degradation characteristics of the five feeds were determined in two sheep.
2.1. Preparation of the experimental feeds
Direct cut silage (DC), silage acidified with formic acid (FA), wilted silage (W), highly wilted silage (HW) and hay (H) were prepared from the grass from a single meadow (primarilyPoa pratensis,Poa trivialis andLolium perenne). The times from cutting to ensiling were 1.8, 1.8, 6.5 and 23 h for DC, FA, W and HW silage, respectively. Hay was harvested 55 h after the cutting. Materials for W, HW and H were turned over once, three and seven times during wilting and drying, respectively. Weather conditions were favorable. Grass was chopped by a precision-chop forage harvester (theoretical chop length 6 mm) and ensiled in concrete experimental silos with a volume of 0.785 m3. Silos were filled manually, herbage was consolidated continuously during filling and sealed immediately thereafter. Each type of silage was prepared in duplicate. FA was preserved by adding 85% formic acid at a rate of 5 kg tÿ1of fresh material. Formic acid was diluted with water in the ratio 1 : 1 and applied by means of a plastic watering can at the filling site. The silos were opened after 103 days. Hay was stored in a barn and passed through the same precision-chop harvester before feeding.
2.2. Experiment 1
2.2.1. Animals and feeding
Four wethers (local breed SolcÏavska) were used. The animals weighed 67.4 kg (SEM 1.9) at the beginning and 77.3 kg (SEM 1.2) at the end of the experiment. They were assigned to a balanced incomplete block design (five dietsfour animalsfive periods). Animals were kept in metabolism cages with free access to fresh water. Animals were fed in two equal meals at 7.00 and 19.00 h. Each experimental period lasted 21 days. From Days 1 to 11 the animals were fed ad libitum. The feed was offered 0.15 in excess of the intake from the previous day. From Days 12 to 21, feeds were offered at 0.9 of the lowest ad libitum intake measured during the first experimental period (58.6 g DM kgÿ1W0.75). Diets were supplemented with 15 g dayÿ1 mineral vitamin mix containing 113 g Ca, 57 g P, 6.6 g Mg, 177 g Na, 6.7 g S, 0.82 g Cu, 3.4 g Zn, 1.4 g Mn, 0.03 g Co, 0.03 g J, 1.3 mg Se, 330,000 i.u. vit. A and 35,000 i.u. D3kgÿ
1 . Voluntary dry matter intake (DMI) was calculated from data of Days 5±11. The feed residues were removed daily and 0.30 was sub-sampled and bulked over the 7-day period for DM determination. Dry matter intake at restricted feeding was measured from Days 15 to 21.
2.2.2. Estimation of intestinal flow of microbial protein
Microbial nitrogen (MN) supply to the animal was estimated using purine derivatives (PD) in urine as a marker. Urine was collected daily from Days 15 to 21. The urine was collected into 1M H2SO4to maintain the pH below 3. In sheep with more concentrated urine about 1 l water was placed in the collection vessels in advance, to prevent
precipitation of uric acid. Daily urine amounts were diluted to 5 l with water, mixed and sampled. Samples (30 ml) were stored atÿ208C.
The MN supply was calculated from the PD excretion as described in Chen et al. (1990a, 1991). Measurements obtained from Days 18±21 were used for calculations.
2.2.3. Measurement of rumen liquid and solid outflow rates
The rumen outflow rates of liquid (kL) and particles (kS) were determined using polyethylene glycol (PEG, MW 4000) and Cr-mordanted hay as markers. The fibre for mordanting was prepared by washing the chopped hay for 1 h in a domestic washing machine using laundry detergent as suggested by UdeÂn et al. (1980). The fibre preparation was then washed with water and acetone and dried at 658C. Fibre was mordanted using the procedure of UdeÂn et al. (1980), modified by cooking the fibre with sodium dichromate for 48 h instead of 24 h.
Sixty gram of Cr-mordanted hay was mixed into the small amount of morning diet on Day 16. One hour later 50 g of PEG diluted in 200 ml of water was administrated into the rumen through the oesophageal catheter. Faecal samples for Cr and PEG determination were collected from faeces collectors approximately 24, 30, 36, 48, 72, 96 and 120 h after feeding Cr-mordanted hay. The exact times of defecation were recorded and used in regression analysis for the calculation of the rumen outflow rates. The outflow rate of liquid phase was calculated from the decline of PEG concentration in the faeces according to Grovum and Williams (1973). Values from samples collected at 24, 30, 36 and 48 h were used for the calculation. The outflow rates of particles were calculated based on Cr concentrations in faecal samples which were collected from 24 to 120 h after administration of Cr mordanted hay, using the model G2G1 according to Moore et al. (1992).
2.2.4. Measurement of pH and ammonia concentration in rumen fluid
Rumen fluid was withdrawn through oesophageal catheter 5 h after the morning feeding. pH was measured immediately. Rumen fluid was then centrifuged at 1250g, supernatant acidified with concentrated H2SO4to give the final pH < 4 and stored frozen until ammonia assay. The ammonia measurement was made twice during the restricted feeding.
2.3. Experiment 2
2.3.1. Animals and feeding
Two sheep (one wether EastfrisianBovsÏka and the ram Merino Landschaf) fitted with rumen cannulae (40 mm diameter) were used. The sheep were given meadow hay ad libitum. The quality of hay was similar to that used in the feeding experiment (939 OM, 114 CP, 540 NDF, 317 ADF, 33.6 ADL, 5.2 Ca, 2.9 P; in g kgÿ1DM). The sheep had free access to water and mineral vitamin licks.
2.3.2. Organic matter and protein degradability
Degradabilities were determined using the nylon bag technique as described by érskov et al. (1980). Fresh samples, equivalent to approximately 3 g DM, were weighed into
nylon bags of the internal size 100 mm75 mm and incubated in the rumen for 3, 6, 12, 24, 48 and 72 h. Bags were made from nylon filter cloth LT 075 (Locker Wire Weawers, Warrington, England) with a pore size of 45±55mm. Each determination was done in two
periods incubating two bags per sheep. The samples of all feeds within the incubation time were incubated in the rumen at the same time. Hay was ground with a laboratory mill using a 5 mm screen, while silages were cut by scissors to a particle size less than 10 mm. After the incubation the bags were first rinsed under running tap water and then washed in domestic washing machine for 20 min. Washing losses were determined by soaking bags with samples for 1 h in hot water (398C) instead of incubating them in the rumen and then washed as described previously. Washing loss was determined in four replicates.
Data of protein and organic matter loss from the bags at different incubation times were fitted to the equationpAfort<t0andpab(1ÿeÿct) fort>t0as suggested by McDonald (1981). The termA in the equation represented washing loss and t0lag time. Potential degradability of protein and organic matter (PDGCP and PDGOM) was calculated as (ab) and insoluble but degradable fractions (BCP and BOM) as (a bÿA). The coefficient c represents degradation rate of the insoluble but potentially degradable fractionB. In the case, wheret00 the effective degradabilities of protein (EDGCP) or organic matter (EDGOM) were calculated as EDGabc= ck(érskov and McDonald, 1979) and in the case where t0> 0 the EDG were calculated as EDGab ceÿ ckt0= ck(McDonald, 1981). The measured particle outflow rate (k) obtained from Section 2.2 was used in calculations.
2.3.3. Calculation of Synchrony index
Synchrony indexIS which described synchrony of crude protein and organic matter degradation in the rumen was calculated according to an equation similar to one proposed by Sinclair et al. (1993). The measured values for the daily ratio of the effective degradable protein to organic matter (y) was used instead of a theoretical value of 25 g rumen degradable N kgÿ1rumen degradable organic matter as suggested by Sinclair et al. (1993).
IS
yÿPn
i1jyÿyij=n
y
Daily ratio of the effective degradable protein to organic matter (y, in g N kgÿ1 degradable organic matter) was calculated from the concentrations of crude protein (CP, in g kgÿ1 DM) and organic matter (OM, in kg kgÿ1 DM) taking into account their effective degradabilities in the rumen.
y CPEDGCP
OMEDGOM
The hourly ratios (yi) of degradable protein to degradable organic matter were
cal-culated as quotients between the hourly quantities of degraded protein and organic matter
as
yi
CP bCPcCP= cCPk eÿ cCPktiÿeÿ cCPkti1
eÿ cCPkti12ÿeÿ cCPkti13
eÿ cCPkti24ÿeÿ cCPkti25eÿ cCPkti36ÿeÿ cCPkti37
OM bOMcOM= cOMk eÿ cOMktiÿeÿ cOMkti1
eÿ cOMkti12ÿeÿ cOMkti13
eÿ cOMkti24ÿeÿ cOMkti25eÿ cOMkti36ÿeÿ cOMkti37
:
This equation takes into account degradation characteristics of organic matter and protein in the rumen (b,c) and measured particle outflow rate (k). It was also considered that diet was given to the animals in two equal meals and that feed from the previous three feedings contributed to the protein and energy supply of rumen microorganisms. Due to a lag phase, the first 3 h after feeding were considered as one period which comprised soluble fractions and fractions which were effectively degraded from the timet0onwards.
2.4. Analytical methods
Dry matter (DM) of hay was determined by drying at 1038C to constant weight and DM of silages by the distillation method described by Dewar and McDonald (1961). Crude protein (CP, N6.25) was analyzed according to the Kjeldahl method (Naumann and Bassler, 1976). For silages, Kjeldahl N was determined in fresh samples to prevent the loss of volatile compounds during drying. Neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) were determined according to Goering and Van Soest (1970). Neutral and acid detergent residues were analysed for N to determine the neutral detergent insoluble nitrogen (NDIN) and acid detergent insoluble nitrogen (ADIN). Hemicellulose and cellulose were calculated as NDFÿADF and ADFÿADL, respectively. Ammonia concentration in feeds was determined by steam distillation as a volatile base (Naumann and Bassler, 1976). The concentrations of lactic, acetic, butyric, propionic and valeric acid in silages were determined using gas chromatography according to Holdeman and Moore (1975). Uric acid, allantoin, xanthine and hypoxanthine (the four compounds collectively referred to as purine derivatives) in urine samples were determined by the autoanalyzer method as described by Chen et al. (1990b). PEG in faeces samples was determined turbidimetrically according to Smith (1958). For determination of Cr concentration a modification of the method which was described by Le Du and Penning (1982) was used. After ashing at 5508C ashes were digested with nitric acid. Concentrations of Cr in digests were determined on Perkin Elmer 2380 Atomic Absorption Spectrometer at 357.9 nm using nitrous oxide-acetylene flame. The high temperature nitrous oxide-acetylene flame was reported to reduce the chemical and matrix interferences which commonly occurs in the air-acetylene flame (Perkin Elmer, 1982).
2.5. Statistical analysis
Statistical analysis was performed with the aid of STATGRAPHICS (1991). Values (20 observations from four animals and five periods) were examined by ANOVA as a
balanced incomplete block design to examine the effect of diet, animal and period (Section 2.2). In the case of degradability characteristics (Section 2.3) which were determined using two sheep in two time replicates, sheep and period were used as a blocks.
3. Results
3.1. Chemical composition of hay and silage
The chemical composition of the silages and hay is given in Table 1. Silage DM content varied from 213 g kgÿ1 in DC to 521 g kgÿ1 in HW and covered the range typical for samples from Slovenian farms (Pen and Kapun, 1997). Although the silages and hay were prepared from the same parental material, they differed in their DM composition. Relatively high concentrations of NDF in H and HW in comparison with DC, FA and W were the consequence of higher hemicellulose fraction while the cellulose fraction was similar. Crude protein concentration was lower in H than in silages. The difference was probably the consequence of particle losses of material during haymaking. In comparison with ensiling, haymaking increased the proportion of NDIN, but not the ADIN. In DC, almost 0.18 of the protein was broken down to ammonia. Concentrations of ammonia N in FA, W and HW were considerably lower than in DC.
Wilting of grass prior to ensiling restricted fermentation in the silo. In W and HW the concentrations of all acids were lower and the pH was higher than in DC (Table 1). In comparison with DC, addition of formic acid only reduced the concentration of butyric acid while the concentration of lactic acid seemed to be unchanged and the concentration of acetic acid even increased. The concentrations of total organic acids in FA, W and HW were equivalent to 76.4, 27.2 and 5.9% of the concentration in DC silage.
3.2. Voluntary feed intake
Voluntary dry matter intake of silages was considerably lower than that of H (Table 2). During the restricted feeding the animals received the same amounts of feed and therefore the differences in realized dry matter intake were not statistically significant.
3.3. Rumen pH, ammonia concentration and outflow rate
Rumen pH was maintained between 6.5 and 6.8 and not affected by the preservation method (Table 3). The sheep given DC had the highest rumen ammonia concentration (Table 4). The rumen ammonia concentrations were somewhat lower in W and HW and the lowest in H. It should be mentioned that rumen fluid samples were taken only 5 h after feeding and therefore the results do not offer complete information on ammonia release in the rumen. Particulate outflow rate varied from 0.039 hÿ1in W to 0.049 hÿ1in FA and liquid outflow rate varied from 0.052 hÿ1in DC to 0.064 hÿ1in W (Table 3). The differences were not significant (P> 0.1).
Table 1
Chemical composition of parental material and corresponding preserved forages (n4) Parental materialb
aDry matter in silages was determined by toluene distillation method. bFor parental materialn5.
3.4. Microbial protein synthesis in the rumen
The effect of the preservation method on the PD excretion and the calculated MN supply is shown in Table 4. Daily PD excretion and calculated MN supply were higher in H and HW than in DC, FA or W, but the differences were not statistically significant (P> 0.05). However, MN kgÿ1 DM intake or per kilogram digestible organic matter intake differed significantly among the five feeds.
Table 3
Influence of diet on rumen pH, ammonia concentration, solid (kS) and liquid (kL) phase outflow rate
Direct cut
184 177 144 145 122 23 <0.1
kS(hÿ1) 0.040 0.049 0.039a 0.041 0.047 0.007 NS
kL(hÿ1) 0.052 0.058 0.064 0.061 0.057 0.011 NS
aCalculated on the basis of data from three sheep.
Table 2
Dry matter intake during ad libitum and restricted feeding regime (g DM dayÿ1)*
Direct cut
Ad libitum feeding 1332a 1386a 1326a 1151a 1749b 134 <0.05
Restricted feeding 1219 1223 1200 1190 1223 64 NS
* a,bMeans with different superscripts are significantly different (P< 0.05).
Table 4
Purine derivative (PD) excretion and calculated microbial nitrogen (MN) supply in sheep as affected by grass preservation method*
Per day 15.68 15.89 15.15 17.44 18.06 1.15 NS
Per kilogram DMI 12.85a 13.12a 12.62a 14.62b 14.78b 0.40 <0.01
MN supply (g)
Per day 13.54 13.72 13.07 15.08 15.61 1.00 NS
Per kilogram DMI 11.09a 11.32a 10.89a 12.63b 12.77b 0.35 <0.01
Per kilogram RDOMI 21.80a 23.78b 22.23ab 28.02d 26.10c 0.74 <0.001
*DMI: Dry matter intake; RDOMI: Rumen degradable organic matter intake, calculated as organic matter
intakeEDGOM/1000, EDGOM as presented in Table 5; a,b,c,d Means with different superscripts are
significantly different (P< 0.05).
3.5. Degradation characteristics in the rumen
The influence of forage preservation method on the characteristics of OM degradability in the rumen is presented in Table 5. The washing loss of the feeds which were wilted on the field (W, HW and H) was lower than in DC and FA. The only feed with considerably lower degradation curve (lower organic matter disappearances) than any of the others was HW. HW also had the lowest washing loss and significantly lower EDGOMthan any of the others.
Preservation method markedly affected protein degradability in the rumen (Table 6). DC expressed the highest readily soluble protein fraction and had the highest protein loss from the nylon bags at all incubation times. Wilting and acidifying grass with formic acid prior to ensiling reduced the soluble protein fraction. In comparison with DC, silages from wilted material had lower degradation rates, whilst formic acid treated silage did not. H had almost two times lower soluble protein fraction than that of any of the silages. Preservation method did not significantly affect the potential protein degradability fraction which varied from 890 g kgÿ1 in H to 915 g kgÿ1 in W. The insoluble but
Table 5
Characteristics which represent the organic matter degradation of different silages and hay from the same parental material in the rumen*
Direct cut
*A: Soluble fraction; B: Insoluble but degradable fraction;c: Coefficient from the exponential equation
pab(1ÿeÿct) representing the degradation rate;t0: Lag time; PDG: Potential degradability (AB); EDG:
Effective degradability calculated using a rumen solid phase outflow rates from the Table 3;a,b,cMeans with different superscripts are significantly different (P< 0.05).
Table 6
Characteristics which represent the protein degradation of different silages and hay from the same parental material in the rumen*
*All terms are defined in Table 5; a,b,c,d Means with different superscripts are significantly different
(P< 0.05).
degradable fraction was closely related to readily soluble protein fraction (r ÿ1.00,
P< 0.001). There were no lag times in the degradation of protein. The fitted a values were in all instances close to the measured soluble protein fraction. Effective protein degradabilities varied from 677 g kgÿ1in H to 855 g kgÿ1in DC. The values for effective protein degradability in Table 6 also comprised, beside the differences in the characteristics of protein degradation, the differences in outflow rates of particles in the rumen (Table 3). The differences in outflow rates only had a minor effect on the differences in effective protein degradability. The latter, which was calculated assuming an average outflow rate of 0.043 hÿ1, only differed from that calculated using individual outflow rate data by 8 g kgÿ1.
3.6. Synchrony index
There were large differences between feeds in their pattern of changes in the hourly ratio of degradable protein to organic mater (Fig. 1). In silages the ratios within the first 3 h after feeding ranged from 40.7 to 53.8 g N kgÿ1EDGOM, but subsequently decreased to a lower level (6.1±10.5 g N kgÿ1 EDGOM). In H the ratio within the first 3 h was considerably lower than in silages (24.7 g N kgÿ1EDGOM) but it maintained relatively unchanged with time. The calculated synchrony index was 0.22±0.34 for the silages and 0.84 for the hay (Table 7).
4. Discussion
4.1. Organic matter and protein degradability in the rumen
The results indicate that wilting of grass prior to ensiling and drying of grass during haymaking decreased the effective organic matter degradability in the rumen. The
Fig. 1. The diurnal variation in ratio between the effectively degraded protein and organic matter in direct cut silage (&), formic acid treated silage (~), wilted silage (*), highly wilted silage (~) and hay (&).
observations are in agreement with those of Thomas et al. (1969); Beever et al. (1971) and Donaldson and Edwards (1976), who found lower apparent whole tract digestibility in wilted silages than in the unwilted. Despite differences in effective organic matter degradability, DC and H did not differ in the effective degradability of non-protein organic matter (0.503 versus 0.500). This indicates that the reduced effective organic matter degradability in hay was completely due to a reduction in protein degradability. However, the reduced effective organic matter degradability in the wilted silages was due to a reduction in degradation of not only protein but also non-protein organic matter (0.489 and 0.444 in W and HW, respectively).
The soluble protein and ammonia N fractions in W, HW and FA were considerably lower than in DC (Tables 1 and 6). The results should be discussed in the light of understanding that protein is broken down to peptides and free amino acids by the action of plant proteases (Kemble, 1956) while the breakdown of amino acids to ammonia and other forms of non-protein N is mainly caused by the action of proteolytic clostridia in the silo (Ohshima and McDonald, 1978). It has been shown that large amounts of ammonia can be produced also by enterobacteria (Henderson, 1985) while lactic acid bacteria have only limited capability to deaminate amino acids (McDonald et al., 1991). Both the action of plant proteases and the growth of clostridia and enterobacteria can be inhibited by reducing pH value or by increasing DM concentration (Macpherson, 1952; Kemble and Macpherson, 1954; Wieringa, 1969; Weise and Hornig, 1975). This may explain why the wilted silages and formic acid treated silage had lower soluble protein and ammonia concentrations. Wilting can also directly affect the protein solubility of grassland forage. The results from Mangan et al. (1991) indicate that a part of soluble protein fraction can be bound on the fibre fraction during wilting.
The effective protein degradability decreased with a high DM concentration in the preserved forage. The results are in agreement with studies on the effect of wilting on the protein degradability of silage (Van Vuuren et al., 1989; Van Vuuren et al., 1990; Campbell and Buchnan-Smith, 1991; Lebzien and GaÈdeken, 1996) although some authors reported that protein degradability in wilted silages can be similar or slightly higher than in unwilted silages (Gordon and Peoples, 1986; Narasimhalu et al., 1989; Teller et al., 1992; Yan et al., 1998). Although based only on a limited number of observations (DC, W, HW and H), the regression EDGCP (g kgÿ1)904ÿ0.26DM (g kgÿ1) was Table 7
Average daily ratio between effectively degradable protein and organic matter and index of synchrony (IS) which
describes a degree of diurnal deviation of the ratio between effectively degradable protein and organic matter from its daily average
aValues are calculated on the basis of degradability characteristics from Tables 4 and 5 taking into account
the particulate outflow rates from Table 3.
significant (P< 0.05,R20.98, SE14). The coefficients of the regression equation are similar to those reported by Tamminga et al. (1992) for 35 silages with DM concentrations ranging from 143 to 673 g kgÿ1 (intercept 907 and slope ÿ0.32 ). In both, the present study and the study reported by Tamminga et al. (1992) protein degradabilities were determined using the in sacco method. The assumption that all the nitrogen which escaped from the nylon bags at 0 h incubation was completely degraded in the rumen may not be true. At least part of it may escape from rumen without being degraded. That would impose overestimation of the protein degradability in feeds with high soluble protein fraction. As a consequence, the differences between feeds contrasting in dry matter concentration would be overestimated as well.
4.2. Microbial protein synthesis in the rumen
Microbial protein supply in HW and H was significantly higher than in DC and W (Table 4). The results support the theoretical assumption performed by Chamberlain (1987) who suggested that extensive fermentation in the silo can reduce the energy for microbial synthesis in the rumen by about 15±20%. They are also in agreement with the findings that wilting of grass prior to ensiling supports a higher microbial protein yield in the rumen (Merchen and Satter, 1983; Narasimhalu et al., 1989; Teller et al., 1992; Yan et al., 1998). However, the estimates of microbial protein synthesis are not consistent and there were also some studies indicating that microbial protein yield in hay can be lower than in silages (Merchen and Satter, 1983; Jaakkola and Huhtanen, 1993) and lower in wilted silages than in unwilted (Thomson and Beever, 1980). The reason for disagreements could derive from variability in materials by means of their suitability for ensiling as well as by the different conditions for wilting and drying.
Similar to wilting, the acidification with formic acid was also expected to increase the microbial protein supply through the inhibition of fermentation in the silo. The response of microbial protein synthesis to the formic acid treatment (Table 4) was lower than expected from the decrease in the concentration of total acids (Table 1) and considerably lower than reported by Jaakkola et al. (1993). Reports from the literature on the effect of formic acid on microbial protein synthesis are conflicting. Chamberlain et al. (1982) found that formic acid did not affect microbial protein synthesis while Hvelplund and Mùller (1976) reported that addition of formic acid even decreased it.
Although not significant (r ÿ0.73, P> 0.1), the negative correlation between the microbial protein supply and the total acids concentration in feedstuffs generally supported the hypothesis that the reduced microbial protein supply in silages was due to the fermentation end-products which did not contribute as a source of energy to the microbial growth in the rumen. However, in wilted silage (W) relatively low microbial protein yield can not be explained by the concentration of total acids. One of the possible explanations for the variability in microbial protein supply could be the degree of synchrony between supply of fermentable energy and degradable protein in the rumen. Microbial protein supply was positively related to the index of synchronyIS(r0.72), however, the correlation was not significant (P> 0.1). In DC and W the lowest IS coincided with the lowest microbial protein supply. Higher IS is expected not only to improve the direct supply of rumen microbes with rumen degradable N but also to
provide some specific compounds which can not be supplied by the rumino-hepatic circle. Sinclair et al. (1993, 1995) found out that by synchronizing the dietary energy and N supply in the rumen microbial N supply can be increased by about 10±20%. Increased efficiency of microbial protein synthesis can be expected only in once or twice a day feeding regime as used in the present experiment. In all-day feeding regimes the problem of asynchronous diet would be eliminated by the permanent food intake. Anyway, the role of synchrony of energy and nitrogen release should be interpreted with caution since its effect can be confounded with any of the other characteristics of feeds, which are important for microbial growth in the rumen.
Efficiency of microbial protein synthesis may also be affected by the rumen digesta outflow rate (Harrison et al., 1975; Kennedy and Milligan, 1978; Dewhurst and Webster, 1992; Murphy et al., 1994) and by the rumen pH value (Strobel and Russell, 1986). In the present experiment, neither the liquid and the solid phase outflow rates (Table 3) nor the rumen pH value (Table 4) were affected by the diet. Therefore, we can conclude that the rumen pH value and outflow rate were not the reason for variability in the microbial protein synthesis in diets based on various preserved forages.
4.3. Model of nitrogen transactions within the digestive tract of the sheep
On the basis of the results on chemical composition, protein degradability in the rumen, microbial protein synthesis and outflow rate of solid phase from the rumen, the model of nitrogen transactions within the digestive tract was developed (Fig. 2). Despite the lower concentration of N, the concentration of insoluble N in H was considerably higher than in any of the silages. Compared to DC the concentration of insoluble N was also increased because of the wilting (W and HW) or acidifying (FA) of grass prior to ensiling. On the other hand, the concentrations of ammonia N and soluble non-ammonia N in hay were more than twofold lower than in the silages. Despite less rumen degradable N from hay compared to silage, more slowly degrading N in hay compensated for this so that greater efficiency of microbial protein synthesis was possible. The N degradability was in general negatively related to the microbial protein synthesis in the rumen. That was the reason for the huge differences in the capture of the rumen degradable protein which was the lowest in DC, considerably higher in FA, W and HW and the highest in H. Capture greater than 1.00 in H indicates that certain amount of endogenous N had to be transferred to the rumen and effectively incorporated into microbial protein. Based on indirect measurements it seems that in silages, especially in DC, considerable losses of ammonia N from the rumen can be expected. This N is assumed to be lost from the animal body mainly via renal route as urea.
The estimated metabolizable protein content of DC, FA, W, HW and H was 0.395, 0.488, 0.514, 0.567 and 0.716 of dietary protein, respectively (Fig. 2). It should be pointed out that badly fermented direct cut silage was compared with silages and hay which were wilted or dried under favourable weather conditions. Therefore, relative large differences which were obtained in the present study can be considered as extreme. In the case of adverse wilting conditions, the protein value of wilted silages and hay could be lower than that of direct cut silage. The importance of fast wilting rate was already exposed by Patterson et al. (1998) and Yan et al. (1998) who found out that the
introduction of an improved wilting system resulted in higher protein value and better utilization of wilted silages for milk production than those recorded in the early wilting studies at the same institute.
5. Conclusions
The results indicate that protein degradability and microbial protein synthesis in the rumen were affected by the method used for the preservation of grassland forage. Protein degradability of hay was lower than in silages, and that of wilted silages lower than the unwilted. Compared to unwilted silages, protein degradability was also reduced by the formic acid treatment prior to ensiling. Compared to unwilted silage, silage wilted to moderate DM and unwilted silage treated with formic acid, the microbial protein
Fig. 2. Model of nitrogen transactions (in g N kgÿ1DM intake) within the digestive tract of sheep given direct
cut silage (DC), formic acid treated silage (FA), wilted silage (W), highly wilted silage (HW) or hay (H). Digestibility of undergraded dietary N was calculated as 0.9(undegraded dietary protein-ADIN)/undegraded dietary protein (AFRC, 1992).
synthesis in hay and highly wilted silage was increased considerably. The protein value of hay was better than in silages and that of the wilted silages better than the unwilted.
Acknowledgements
Research was financed by the Slovenian Ministry for Science and Technology. Collaborative work between Agricultural Institute of Slovenia and Rowett Research Institute was supported by The British Council.
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