Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol82.Issue3-4.Dec1999:

Animal Feed Science and Technology
82 (1999) 195±212

The effect of method of forage preservation on
the protein degradability and microbial
protein synthesis in the rumen
J. VerbicÏa,*, E.R. érskovb, J. ZÏgajnarc, X.B. Chenb,
Vida ZÏnidarsÏicÏ-Pongraca
a

Agricultural Institute of Slovenia, 1000 Ljubljana, Hacquetova 17, Slovenia
b
Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK
c
University 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 ˆ 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 kgÿ1 for 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 to IS (r ˆ 0.72, P > 0.1). Estimated metabolizable protein concentrations
were 53.4, 59.9, 57.1, 70.3 and 75.3 g kgÿ1 DM 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

*

Corresponding author. Tel.: ‡386-61-1375375; fax: ‡386-61-1375413

E-mail address: joze.verbic@kis-h2.si (J. VerbicÏ)
0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 1 0 2 - 9

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J. VerbicÏ et al. / Animal Feed Science and Technology 82 (1999) 195±212

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

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197

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
(primarily Poa pratensis, Poa trivialis and Lolium 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 tÿ1 of 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 diets  four animals  five
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 kgÿ1 W0.75). 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. D3 kgÿ1.
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 H2SO4 to 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

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J. VerbicÏ et al. / Animal Feed Science and Technology 82 (1999) 195±212

precipitation of uric acid. Daily urine amounts were diluted to 5 l with water, mixed and
sampled. Samples (30 ml) were stored at ÿ208C.
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 H2SO4 to 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 Eastfrisian  BovsÏka 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 kgÿ1 DM). 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

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199

nylon bags of the internal size 100 mm  75 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±55 mm. 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 equation p ˆ A for t < t0 and p ˆ a ‡ b (1 ÿ eÿct) for t > t0 as suggested
by McDonald (1981). The term A in the equation represented washing loss and t0 lag
time. Potential degradability of protein and organic matter (PDGCP and PDGOM) was
calculated as (a ‡ b) and insoluble but degradable fractions (BCP and BOM) as (a ‡
b ÿ A). The coefficient c represents degradation rate of the insoluble but potentially
degradable fraction B. In the case, where t0  0 the effective degradabilities of protein

(EDGCP) or organic matter (EDGOM) were calculated as EDG ˆ a ‡ bc=…c ‡ k† (érskov
and McDonald, 1979) and in the case where t0 > 0 the EDG were calculated as
EDG ˆ a ‡ b c eÿ…c‡k† t0 =…c ‡ k† (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 index IS 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 kgÿ1 rumen degradable organic matter as suggested by Sinclair et al.
(1993).
IS ˆ

yÿ

Pn

jyÿyi j=n
y

iˆ1

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.
yˆ

CPEDGCP
OMEDGOM

The hourly ratios (yi) of degradable protein to degradable organic matter were calculated as quotients between the hourly quantities of degraded protein and organic matter

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J. VerbicÏ et al. / Animal Feed Science and Technology 82 (1999) 195±212

as
 


CP…bCP cCP †=…cCP ‡ k† eÿ…cCP ‡k†ti ÿeÿ…cCP ‡k†ti‡1 ‡ eÿ…cCP ‡k†ti‡12 ÿeÿ…cCP ‡k†ti‡13

 

‡ eÿ…cCP ‡k†ti‡24 ÿeÿ…cCP ‡k†ti‡25 ‡ eÿ…cCP ‡k†ti‡36 ÿeÿ…cCP ‡k†ti‡37


 
yi ˆ
OM…bOM cOM †=…cOM ‡k† eÿ…cOM ‡k†ti ÿeÿ…cOM ‡k†ti‡1 ‡ eÿ…cOM ‡k†ti‡12 ÿeÿ…cOM ‡k†ti‡13

 

:
‡ eÿ…cOM ‡k†ti‡24 ÿeÿ…cOM ‡k†ti‡25 ‡ eÿ…cOM ‡k†ti‡36 ÿeÿ…cOM ‡k†ti‡37
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 time t0 onwards.
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, N  6.25) 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

J. VerbicÏ et al. / Animal Feed Science and Technology 82 (1999) 195±212

201

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 kg ÿ1 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 hÿ1 in W to 0.049 hÿ1 in
FA and liquid outflow rate varied from 0.052 hÿ1 in DC to 0.064 hÿ1 in W (Table 3). The
differences were not significant (P > 0.1).

202

Dry mattera
Organic matter
NDF
ADF
ADL
Hemicellulose
Cellulose
Crude protein
NDIN
ADIN
NH3±N
PH
Lactic acid
Acetic acid
Butyric acid
Propionic acid
Valeric acid
Total acids
a
b

g kgÿ1
g kgÿ1
g kgÿ1
g kgÿ1
g kgÿ1
g kgÿ1
g kgÿ1
g kgÿ1
g kgÿ1
g kgÿ1
g kgÿ1

DM
DM
DM
DM
DM
DM
DM
total N
total N
total N

g kgÿ1
g kgÿ1
g kgÿ1
g kgÿ1
g kgÿ1
g kgÿ1

DM
DM
DM
DM
DM
DM

Parental materialb
x  SEM

Direct cut silage
x  SEM

Formic acid treated
silage x  SEM

Wilted silage
x  SEM

Highly wilted
silage x  SEM

Hay x  SEM

ÿ
931  4
524  12
307  9
38  3
217  4
269  6
132  6
±
±
±
±
±
±
±
±
±
±

213  5
921  5
527  4
341  1
30  2
186  4
311  2
135  3
92  5
71  6
179  26
4.2  0.1
75.6  7.1
11.0  1.7
27.1  4.2
1.04  0.30
0.12  0.03
114.8  8.7

236  12
923  5
506  6
329  10
29  2
177  5
300  9
123  4
102  7
65  2
81  23
4.0  0.1
64.9  13.5
16.3  3.0
6.2  1.9
0.30  0.09
0.02  0.01
87.7  13.8

432  5
931  2
522  9
323  6
30  2
199  5
293  6
111  2
94  2
69  4
54  18
4.7  0.3
18.5  2.7
3.6  0.5
8.9  1.4
0.12  0.01
0.02  0.01
31.8  4.3

521  7
931  1
551  7
338  4
31  2
214  5
307  5
124  3
109  11
68  2
38  12
5.6  0.2
2.4  0.4
4.3  0.5
0.1  0.0
0.03  0.01
0.00  0.00
6.8  0.6

893  3
936  3
541  3
315  3
27  1
256  1
288  3
105  2
183  8
65  2
21  0
±
±
±
±
±
±
±

Dry matter in silages was determined by toluene distillation method.
For parental material n ˆ 5.

J. VerbicÏ et al. / Animal Feed Science and Technology 82 (1999) 195±212

Table 1
Chemical composition of parental material and corresponding preserved forages (n ˆ 4)

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J. VerbicÏ et al. / Animal Feed Science and Technology 82 (1999) 195±212
Table 2
Dry matter intake during ad libitum and restricted feeding regime (g DM dayÿ1)*

Ad libitum feeding
Restricted feeding
* a,b

Direct cut
silage

Formic acid
treated silage

Wilted
silage

Highly wilted
silage

Hay

SED
DF 8

Sig.

1332a
1219

1386a
1223

1326a
1200

1151a
1190

1749b
1223

134
64