Use of Nylon Bag Technique to Determine Nutritive Value and Degradation
Kinetics of Iranian Alfalfa Varieties
Amir Reza Safaei,
The aim of the study was to determine the chemical composition and ruminal
degradation characteristics of two alfalfa varieties including Kareyonge
(KAR) and Hamedani (HAM) grown in West Azerbaijan in Iran, using in
situ technique. The nutritional parameters were Dry Matter (DM), Organic
Matter (OM) and Crude Protein (CP). Nylon bags filled with 5 g of each
forage were suspended in the rumen of three cannulated Gezel rams immediately
before feeding and incubated for 7 different times (0, 4, 8, 16, 24, 48
and 72 h). Parameters for degradation kinetics included readily degraded
fraction, slowly degraded fraction, lag time and fractional rate of passage.
No significant difference found between DM, CP, Ash and Ether Extract
(EE) of two alfalfa varieties although the difference for Crude Fiber
(CF), Neutral Detergent Fiber (NDF) and Acid Detergent Fiber (ADF) were
significant (p<0.01).The degradability of DM, OM, CP and Effective
Rumen Degradable Protein (ERDP) at a rate of 0.05/h for KAR and HAM varieties
were 54.2, 52.16, 40.1%, 72.57 g kg-1 DM and 56.97, 54.9, 39.25%,
89.4 g kg-1 DM, respectively. Calculations based on in situ
degradability indicate that HAM alfalfa can have a higher inclusion than
KAR alfalfa in diets for ruminants because of lower NDF, greater cell
contents and ERDP.
to cite this article:
Afshar Mirzaei-Aghsaghali, Naser Maheri-sis, Ali Mirza-Aghazadeh, Amir Reza Safaei, Amir-Farhang Houshangi and Abolfazl Aghajanzadeh-Golshani, 2008. Use of Nylon Bag Technique to Determine Nutritive Value and Degradation
Kinetics of Iranian Alfalfa Varieties. Asian Journal of Animal and Veterinary Advances, 3: 214-221.
Alfalfa, harvested as hay, is an important forage crop for ruminants
because of its high protein concentration; however, research has indicated
that the proteins in alfalfa are highly susceptible to degradation during
field-wilting and ruminal fermentation (Coblentz et al., 1997).
Protein requirement of animals was used to be calculated based on crude
protein concentrations of diets. However, studies have shown that addition
of escape protein of fast growing ruminants and high producing dairy cows
have resulted in an improvement in animal performance, indicating that
crude protein system is lacking in term of meeting the protein requirements
of animals. Therefore, metabolizible protein system was introduced to
more accurately and preciously meet the protein requirements of ruminant
animals. Forage protein serves as a source of metabolizable protein to
the ruminants by providing both ruminally degradable protein for microbial
growth and some ruminally undegradable protein for intestinal digestion.
Due to rapid and extensive degradation of forages in the rumen, escape
protein concentration of forages are usually low. Many factors such as
maturity, forage species and preservation method influence the ruminal
degradability of forage CP content. The analytical procedures for feedstuffs
so as to characterize nutrient composition have been standardized.
Recent improvements clarified specific constituents of macronutrients.
For instance, the use of degradable and undegradable rumen fractions in
place of crude protein is done to improve the utilization of feedstuffs.
The nylon bag technique offers an easy, fast, economical and effective
method to determine Crude Protein (CP) fractions (NRC, 1989; Hoffman
et al., 1993; Broderick, 1995; Kaya et al., 2004).
In Lucerne, Broderick and Buxton (1991) found variation in protein degradability
among 19 M. sativa accessions and 3 M. falcata accessions.
Cherney et al. (1992) demonstrated that stage of maturity affects
rate of ruminal NDF degradation so, degradation kinetics of forages vary.
Bald et al. (1993) investigated degradation kinetics of alfalfa
at early bud, early bloom and full bloom stages and showed that the digestibility
of DM and CP linearly decreased with advancing stage of maturity. Skinner
et al. (1994) found difference in degradation rates of proteins
among nine Lucerne accessions. Kaya et al. (2004) also found cumulative
disappearance and effective degradability for DM, OM, CP and CF linearly
decreased with advancing maturity.
Alfalfa hay is an important forage crop for ruminants. There are two
common varieties of Lucerne in Iran including Hamedani and Kareyonge.
Inclusion of Hamedani in ruminant diets is higher than that of Kareyonge.
A little study has been determined some nutritional characteristics of
alfalfa varieties in Iran (Maheri-sis et al., 2007).
This study investigated the differences between chemical composition
and in situ degradation kinetics of Hamedani (HAM) and Kareyonge
(KAR) hays for total DM, OM and N.
MATERIALS AND METHODS
Two Iranian common alfalfa varieties (Hamedani and Kareyonge) were
used in the experiment. Samples of two alfalfa hays were collected in
2005, from ten farms, near West Azerbaijan, Iran (located in the Urmia
and Miandoab city) and evaluated at the laboratories of Islamic Azad University-Shabestar
Branch. Both alfalfas, at harvest, were estimated to be at late maturity
(both varieties in mid to late bloom). Samples were collected, oven-dried
at 60°C for 48 h, ground (5 mm screen) and prepared for in situ
analysis (Andrighetto et al., 1993).
Dry Matter (DM) was determined by drying the samples at 105°C overnight
and ash by igniting the samples in muffle furnace at 525°C for 8 h.
Nitrogen (N) content was measured by the Kjeldahl method (AOAC, 1990).
Crude Protein (CP) was calculated as Nx6.25. Neutral detergent fiber,
ADF, acid-detergent sulfuric acid (ADL) and Acid-Insoluble Ash (AIA) were
determined by procedures outlined by Goering and Van Soest (1970) with
modifications described by VanSoest et al. (1991), sulfite was
omitted from NDF analysis. Hemicellulose and cellulose were calculated
as NDF-ADF and ADF-ADL-AIA, respectively (Andrighetto et al., 1993).
In situ Degradation Procedures
Three hundred fifty five kilogram ruminally cannulated Gezel rams
were used to determine in situ degradation characteristics. Rams
were housed in individual tie stalls bedded with sawdust. Rams fed ground
alfalfa hay containing 14% CP and 45% NDF were used for incubation of
samples in Dacron bags in this study. Alfalfa hay was offered 1.25 x maintenance
levels of rams (Karsli et al., 2002).
In situ procedures were the same as those described previously
(Coblentz et al., 1997), Dacron bags (18x9 cm; 520 mm pore size)
were filled with 5 g samples of dried ground forage. Suspension of bags
in the rumen was accomplished by tying of bags, into tygon tubing with
nylon string. Sample in Dacron bags were placed in the rumen of rams and
incubated for the periods of 0, 4, 8, 16, 24, 48, 72 h. After the removal
of bags from the rumen, bags were washed in cold water until rinse were
clear and dried at 60°C for 48 h (Karsli and Russell, 2002). Remaining
residues were analyzed for DM, OM and N concentrations.
Dry matter, organic matter and N were divided into three fractions as
follows: 1) The soluble DM, OM or N fraction (fraction a) determined as
DM, OM or N loss during the washing process, 2) The potentially digestible
DM, OM or N fraction (fraction b) determined as the differences between
initial DM, OM or N content after washing and the amounts of DM, OM or
N recovered after a 72 h incubation, 3) The indigestible fraction (fraction
c) determined as the amount of DM, OM or N residue recovered after a 72
h incubation (Karsli and Russell, 2002).
Rumen degradation kinetics for DM, OM and CP were calculated using the
nonlinear model proposed by Ørskov and McDonald (1979):
|| Percentage of degradability for response variables
||Time relative to incubation (h)
||Highly soluble and readily degradable fraction
||Insoluble and slowly degradable fraction
||Rate constant for degradation
||2.7182 (Natural logarithm base)
Following determination of these parameters, the effective degradability
of nutrients in the two varieties was calculated using and equation described
by Ørskov and McDonald (1979):
||Effective degradability for response variables (%)
|| Highly soluble and readily degradable fraction
|| Insoluble and slowly degradable fraction
||Rate constant for degradation
||Rate constant of passage
When calculating effective degradability, rate constant of passage was assumed
to be 0.02, 0.05 and 0.08% per hour (Bhargava and Ørskov, 1987) so
that the results could be extrapolated to other ruminants that differ in
All of the data were analyzed by using software of SAS (1985) and
means (obtained from three homogen samples) were separated by independent-samples
t-test (Steel and Torrie, 1980). All of the means obtained from three
RESULTS AND DISCUSSION
Figure 1 and Table 1 show the differences in the composition
between the two alfalfa varieties. There was considerable variation between
alfalfa hays in term of chemical composition. Cell wall contents (NDF
and ADF), which represent the most important fraction of dry matter for
alfalfa hays, ranged from 43.3 to 49% and 29.4 to 34.2%, respectively.
CP, EE and Ash concentrations for KAR and HAM hays were similar (p>0.05).
||Comparison of feed fractions of Hamedani (HAM) and Kareyonge (KAR)
hays. ADL = Acid-Detergent Lignin, CC = Cell Contents, CE = Cellulose,
HE = Hemicellulose and AIA = Acid-insoluble ash
||Chemical composition of Hamedani (HAM) and Kareyonge (KAR) hays
1: Lignification index based
on NDF, 2: Lignification index based on ADF, SEM: Standard
Error of Means, **: p<0.01; ***: p<0.001
As expected, the concentrations of neutral detergent fiber and ADF were
significantly lower (p<0.001) in HAM hay than KAR hay (Table 1
concentrations of NDF and ADF for HAM hay were in agreement with the results
of Coblentz et al
. (1998). The lignification index (Table 1
either an NDF or an ADF basis (Van Soest, 1982), was similar in KAR and
HAM hays (p>0.05). The concentrations of cellulose and hemicellulose
were significantly higher (p<0.001) in KAR hay than HAM hay (Table 1
This is the first study establishing the rumen degradation characteristics
of these alfalfa varieties in around West Azerbaijan. Although there are
some studies investigating the chemical composition of the KAR and HAM
hays in the region, in none of these studies were degradation characteristics
in rumen taken into account. However, studies have shown that vegetation,
soil type and climate might dramatically affect the utilization of nutrients
by animals (Kaya et al., 2004). The results of this study might
therefore contribute to our current knowledge allow us to find strategies
to improve animal nutrition in the region.
The degradation kinetics of different feed fractions of HAM and KAR hays
are described in Fig. 2 and the corresponding effective degradability
values at three ruminal passage rates are in Table 2. In this study, proportions
of fraction A and B were similar (p>0.05) for hays. Undegradabed fraction
of DM and CP were significantly higher for KAR than (33.5 vs. 27.5 and
48.3 vs. 34.2, respectively). Maximum extents of DM and CP for HAM were
greater (72.43 and 65.8, respectively; p<0.05). However, extents of
degradation of OM for KAR (65.8%) and HAM (67.5%) were similar (p>0.05).
Differences between lag time of DM and OM for HAM and KAR hays (0.8 vs.
1 and 0.83 vs. 0.4, respectively) were non-significant (p>0.05), but
in HAM hay, CP had longer lag time (1.55 vs. 0.25). Effective DM degradability,
at a ruminal turnover rate of 5%/h was 56.98% for HAM hay compared with
54.2% for KAR hay (p<0.01). Effective OM and CP degradability, at ruminal
turnover rates of 2, 5 and 8%/h were similar for HAM and KAR hays (p>0.05).
||Comparison of in situ degradation kinetics of alfalfa hays
||In situ DM, OM and CP degradation characteristics of the
alfalfa hays by sheep
1: KAR = Kareyonge, HAM = Hamedani, 2:
a = Immediately soluble fraction, b = Fraction degradable at a measurable
rate, c = Undegraded fraction and maximum extent = 100 –c,
3: Effective degradability at three ruminal
passage rates, 4: Standard Error, 5: Non-determined,
6: Rate of degradation, *: p<0.05; **: p<0.01
||Effective degradability (%), UDP and ERDP (g k-1g DM)
CP of KAR and HAM hays with different outflow rates
|1: KAR = Kareyonge, HAM = Hamedani, 2:
Effective degradability (ED), 3: Undegradable protein (UDP)
and 4: Effective Rumen Degradable of Protein (ERDP) are
calculated using the equations of AFRC (1993), *: p<0.05; **: p<0.01
CP undegradability, at a ruminal turn over rate of 8%/h was 69.7 g kg-1
DM for HAM hay compared with 51.07 g kg-1
MD for KAR hay (Table 3
, p<0.05). Effective rumen degradability protein, at ruminal turnover
rates of 2, 5%/h were significantly higher (p<0.05) in HAM hay than KAR
hay (Table 3
The percentage of water soluble fraction (fraction a) and potentially
digestible dry matter fraction (fraction b) were similar (p>0.05) and
indigestible dry matter fraction (fraction c) was greater in KAR hay compared
with HAM hay (p<0.05; Table 3). The disappearance of DM, OM and CP
increased with time of incubation in the rumen (4-48 h, Fig. 2). These
values were in line with those of Komprda et al. (1993). They incubated
Lucerne (Medicago sativa), harvested at different stages of maturity,
for 48 h and showed that the disappearance of OM decreased linearly by
up to advancing maturity. They also observed a 22% reduction in CP disappearance
with advancing stage of maturity (Komprda et al., 1993). Decreases
in degradation could be attributed to an increased lignification process
in the cell wall, because lignified tissues limit feed intake and occupy
space in the rumen, which may in turn reduce the attachment of bacteria
to substrates (Kaya et al., 2004).
Overall, the cumulative disappearance pattern for nutrients appears to
decrease linearly with advancing maturity, but slight differences in cumulative
disappearance reported in the literature could be due to differences in
forage sources, stage of maturity and environmental conditions (Kaya et
In the current study, rapidly degradable fraction (a) and slowly degradable
fraction (b) of DM were similar in alfalfa hays (Table 2). These values
were in line with those of Seker (2002). The mean values obtained for
DM potential degradability in the HAM hay (72.43%) is similar to that
obtained by Bald et al. (1993) for alfalfa (77.2%). The slight
differences between the current study and those of reported by Andrighetto
et al. (1993) and Kamalak et al. (2005) might be due in
part to the different plant species used.
Rapidly (a) and slowly (b) degradable fractions of OM were similar in
two varieties. Karsli et al. (2002) found that mean (a), (b) and
(c) values of OM were 28.4, 43.3 and 28.3, respectively for alfalfa hay.
The values obtained in the current study for HAM and KAR hays were also
in line with those of Karsli et al. (2002).
Microbial protein contributes a large amount to the CP that passes to
the intestine of the dairy cow, because microbial CP synthesis in the
rumen is highly dependent on the amount of rumen-degradable OM (Kamalak
et al., 2005).
Rapidly (a) and slowly (b) degradable fractions of CP observed in the
study for HAM hay were in range of result reported for alfalfa by Coblentz
et al. (1998). The difference between the current study and those
of reported by Michalet-Doreau and Ould-Ban (1992) and Elizalde et
al. (1999) might be due in chemical compositions, CP extent and harvesting
Lag time (which indicates the time required for initiation of degradation)
for degradation of CP was significantly higher (p<0.05) in HAM hay.
Lag time may be caused by the need for chemical or physical alteration
of fiber before bacterial attachment and enzymatic digestion can occur,
or by a need for bacterial growth and increases in enzyme content. Physical
factors such as wettability of the substrate, rate of solution and nutrient
limitations also could influence lag time (Olubobokun et al., 1990).
In an overall conclusion, it seems that regarding chemical composition
and degradation kinetics, nutritive value of Hamedani hay was higher than
that of Kareyonge and we can recommended that Hamedani hay may be used
more than Kareyonge hay in ruminant diets.
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