Current Research Journal of Biological Sciences 4(4): 350-354, 2012
ISSN: 2041-0778
© Maxwell Scientific Organization, 2012
Submitted: September 02, 2011
Accepted: October 07, 2011
Published: July 10, 2012
Effects of Zataria multiflora extract as Rumen Modifiers Using in vitro
Gas Production Technique
Mohammad Salamat Azar, Ramin Salamat Doust-Nober, Naser Maheri Sis,
Habib Aghdam Shahryar and Yahya Asadi
Department of Animal Science, Shabestar Branch, Islamic Azad University, Shabestar, Iran
Abstract: This experiment was to determine effects of tow doses (0 and 0.15 mL/30 mL buffered rumen fluid)
of Zataria multiflora extract on Soybean Meal (SBM) degradability were studied by in vitro gas producing
techniques. Gas production test with mixtures of filtered rumen liquid of three taleshi native male cattle rumen
in times of 2, 4, 6, 8, 12, 24, 48, 72 and 96 were performed. The gas volumes for soybean meal in different
incubation times were higher than that of Zataria multiflora extract (0.15 mL/30 mL buffered rumen fluid).
Chemical composition for dry matter, crude protein and Non-Fibrous Carbohydrate were 94.07, 40 and 29.36%,
respectively. The results showed that gas volume at 48 h incubation (for 200 mg dry samples), were 62.12 and
38.01 mL/200 mg DM for soybean meal (control) and Zataria multiflora extract (0.15 mL/30 mL buffered
rumen fluid), respectively. the gas production from soluble fraction (a), the gas production from insoluble
fraction (b), rate constant of gas production during incubation (c) and the potential gas production (a + b)
contents of soybean meal (control) were 4.42 (mL/200 mg DM), 67.1 (mL/200 mg DM), 0.113 (mL/h) and
71.52 (mL/200 mg DM), respectively, the gas production from soluble fraction (a), the gas production from
insoluble fraction (b), rate constant of gas production during incubation (c) and the potential gas production (a
+ b) contents for Zataria multiflora extract (0.15 mL/30 mL buffered rumen fluid) were 3.52 (mL/200 mg DM),
45.54 (mL/200 mg DM), 0.07 (mL/h) and 49.06 (mL/200 mg DM), respectively. Gas volume at 72 h incubation
(for 200 mg dry samples), of soybean meal and Zataria multiflora extract (0.15 mL/30 mL buffered rumen
fluid) were 62.91 and 41.62, respectively. Gas volume at 96 h incubation (for 200 mg dry samples), of soybean
meal and Zataria multiflora extract (0.15 mL/30 mL buffered rumen fluid) were 63.72 and 42.82, respectively.
Keywords: Gas production technique, insoluble fraction, soluble fraction, soybean meal, taleshi native male
cattle, Zataria multiflora extract,
animal, respectively. However, this symbiotic relationship
has energy losses of methane (CH4) and protein losses of
ammonia Nitrogen (N) inefficiencies (Van Nevel and
Demeyer, 1988; Calsamiglia et al., 2007). Methane
produced during anaerobic fermentation in the rumen
represents a 2 to 12% of gross energy loss to the host
animal and contributes to emissions of greenhouse gases
into the environment (Moss, 1993; Bunthoeun, 2007). The
public concern over the routine use of antibiotics and
growth promoters in livestock production has increased
recently because of the risk of the antibiotic residues
presence in milk and meat and its effect on human health.
These led to its prohibition in the European Union in 2006
in animal feeding. Accordingly, there is greater interest in
using plants and plant extracts as alternatives to feed
antibiotics to manipulate ruminal fermentation, improve
feed efficiency and animal productivity. Many plants
produce secondary metabolites such as phenolic
compounds, essential oils and sarsaponins (Calsamiglia
et al., 2006, 2007; Sallam et al., 2009) that affect was
microbial activity (Sallam et al., 2009). The purposes of
this study were to evaluate effects of Zataria multiflora
extract (0.15 mL/30 mL buffered rumen fluid) on gas
INTRODUCTION
Several methods such as in vivo, in situ and in vitro
techniques have been used in order to evaluate the
nutritive value of feedstuffs (Maheri-Sis et al., 2008). The
in vitro gas production technique has proved to be a
potentially useful technique for feed evaluation (Menke
and Steingass, 1988; Getachew et al., 2004; Maheri-Sis et
al., 2008), as it is capable of measuring rate and extent of
nutrient degradation. In addition, in vitro gas production
technique provide less expensive, easy to determine
(Getachew et al., 2004; Maheri-Sis et al., 2008) and
suitable for use in developing countries (Chumpawadee
et al., 2005; Maheri-Sis et al., 2007; Maheri-Sis et al.,
2008). This method also predicts feed intake, digestibility,
microbial nitrogen supply and amount of short chain fatty
acids, carbon dioxides and metabolizable energy of feed
for ruminants (Babayemi, 2007; Maheri-Sis et al., 2008).
Ruminants establish a symbiotic relationship with rumen
microorganisms by which the animal provides nutrients
and optimal environmental conditions for the fermentation
of feeds and microorganisms degrade fiber and synthesize
microbial protein as an energy and protein supply for the
Corresponding Author: Mohammad Salamat Azar, Department of Animal Science, Shabestar Branch, Islamic Azad University,
Shabestar, Iran
350
Curr. Res. J. Biol. Sci., 4(4): 350-354, 2012
volume (mL/200 mg DM) and estimated parameters of
soybean meal at different incubation times using in vitro
gas production technique.
daily with a diet containing alfalfa hay (60%) and
concentrate (40%). The samples were incubated in the
rumen fluid in calibrated glass syringes following the
procedures of (Menke and Steingass, 1988; Menke et al.,
1979) as follows. 200 mg dry weight of the sample was
weighed in triplicate into calibrated glass syringes of
100 mL in the absence and presence of Zataria multiflora
extract (0.15 mL/30 mL buffered rumen fluid). The
syringes were pre-warmed at 39ºC before injecting 30 mL
rumen fluid-buffer mixture into each syringe followed by
incubation in a water bath at 39ºC. The syringes were
gently shaken 30 min after the start of incubation and
every hour for the first 10 h of incubation. Gas production
was measured as the volume of gas in the calibrated
syringes and was recorded before incubation 2, 4, 6, 8, 12,
24, 48, 72 and 96 h after incubation. All samples were
incubated in triplicate with three syringes containing only
rumen fluid-buffer mixture (blank). The net gas
productions for soybean meal samples were determined
by subtracting the volume of gas produced in the blanks.
Cumulative gas production data were fitted to the model
of Ørskov and McDonald (1979):
MATERIALS AND METHODS
Chemical analysis: Dry Matter (DM) was determined by
drying the samples at 105ºC overnight and ash by igniting
the samples in muffle furnace at 525ºC for 8 h and
Nitrogen (N) content was measured by the Kjeldahl
method (AOAC, 1990). Crude protein (CP) was
calculated as N×6.25 (Van Soest et al., 1991). NonFibrous Carbohydrate (NFC) is calculated using the
equation of (NRC, 2001), NFC = 100 (NDF + CP + EE +
Ash). All chemical analyses were carried out in triplicate.
Zataria multiflora samples: During summer season
Zataria multiflora samples were collected from different
parts of Isfahan province. Next, there were drying for one
week and homogeneous mixture were papered for
nutritive chemical analyzes. For determination of (Zataria
multiflora extract) effects, we added Zataria multiflora
extract with dose Zataria multiflora extract (0, 0.15 mL:
200 mg sample) into gas test syringes. All samples were
then ground in a laboratory mill through a 1 mm screen.
P = a+b (1-eGct)
where, P is the gas production at time t, a gas production
from soluble fraction (mL/200 mg DM), b the gas
production from insoluble fraction (mL/200 mg DM), c
the gas production rate constant (mL/h), a+b the potential
gas production (mL/200 mg DM) and t is the incubation
time (h).
Procedure of Zataria multiflora extract preparation:
The Zataria multiflora extract were prepared according to
(Patra et al., 2006; Sallam et al., 2009) with some
modifications. The Zataria multiflora materials were
dried at 50ºC and ground in mills to pass a 1 mm sieve
and 100 g placed in 1000 mL of methanol solvent. The
flasks of all the solvents were stoppered and agitated with
a magnetic stirrer for 24 h at room temperature. Then the
solutions were centrifuged at 3000 g for 10 min. The
residue was re-extracted with 500 mL of methanol for 24
h stirring at room temperature and centrifuged again at
3000 g for 10 min. The Zataria multiflora extract were
combined. Distilled water was evaporated from the
solution at approximately 65ºC by using a rotaryevaporator (Patra et al., 2006; Sallam et al., 2009).
Statistical analysis: Data on apparent gas production
parameters were subjected to one-way analysis of
variance using the analysis of variation model ANOVA of
SAS (2000). Multiple comparison tests used Duncan’s
multiple- t-test (1980). Mean differences were considered
significant at (p<0.05). Standard errors of means were
calculated from the residual mean square in the analysis
of variance. All data obtained from three replicates n = 3.
RESULTS
Treatments and experimental design: The different
doses of Zataria multiflora extract were added to the diet
sample. Tow doses (0, 0.15 mL/30 mL buffered rumen
fluid) of Zataria multiflora extract were investigated as
follow:
C
C
Chemical composition: The chemical composition of
soybean meal shown was in Table 1.
Chemical composition of soybean meal including
Dry matter (DM), crude protein CP) and Non-Fibrous
(Carbohydrate )NFC) were estimated; 94.07, 40 and
29.36%, respectively.
No additive (control);
Zataria multiflora extract (0.15/30 mL buffered
rumen fluid).
In vitro gas production: Gas production volumes
(mL/200 mg DM) at differents incubation times of
soybean meal and Zataria multiflora extract (0.15 mL/30
mL buffered rumen fluid) shown were in Fig. 1.
In vitro gas production: Fermentation of soybean meal
samples were carried out with rumen fluid was obtained
from three fistulated Taleshi native male cattle fed twice
351
Curr. Res. J. Biol. Sci., 4(4): 350-354, 2012
70
Gas volumes (mL/200mg DM)
contents for Zataria multiflora extract (0.15/30 mL
buffered rumen fluid) were 3.52 (mL/200 mg DM), 45.54
(mL/200 mg DM), 0.07 (mL/h) and 49.06 (mL/200 mg
DM).
No additive (control)
Zataria multiflora extract
dose 0.15
60
50
DISCUSSION
40
30
In vivo, in situ and in vitro methods have been used
to evaluate the nutritive value of feedstuffs (Maheri-Sis et
al., 2011). The in vitro gas production technique has
proven to be a potentially useful technique to evaluate the
nutritive value of feedstuffs, since it gives an estimate of
the potential rate and extent of nutrient fermentation in the
rumen. However, this technique is measuring gas
produced by the fermentation of energy containing
components in feeds and not only that of protein (MirzaeiAghsaghali and Maheri-Sis, 2008a; Mirzaei-Aghsaghali
et al., 2008; Maheri-Sis et al., 2007, 2008). Gas volume
at 24 h incubation (for 200 mg dry samples), soluble
fraction (a), insoluble but fermentable fraction (b),
potential gas production (a + b) and rate constant of gas
production (c) of sunflower meal were 42.40, 3.607,
49.32, 52.92 mL/200 mg DM and 0.135 mL/h, for Zataria
multiflora water extract (0.15 mL/30 mL buffered rumen
fluid) 41.41, 3.921, 49.08, 53.004 and 0.135 while for
Zataria multiflora water extract (0.3 mL/30 mL buffered
rumen fluid) were 40.52, 4.655, 48.66, 53.321 mL/200 mg
DM and 0.134 mL/h, respectively (Salamat azar et al.,
2011). Gas volume at 48 h incubation (for 200 mg dry
samples), of sunflower meal, Zataria multiflora water
extract (0.15 and 0.3 mL/30 mL buffered rumen fluid)
were 44.99, 44.49 and 43.27 (mL/200 mg DM),
respectively. Gas volume at 72 h incubation (for 200 mg
dry samples), of sunflower meal, Zataria multiflora water
extract (0.15 and 0.3 mL/30 mL buffered rumen fluid)
were 45.75, 45.23 and 44.25, respectively (Salamat Azar
et al., 2011). These results are in agreement with the
findings of (Salamat Azar et al., 2011). Compounds with
phenolic structures, such as thymol, are more effective as
antimicrobials in comparison with other nonphenolic
secondary plant metabolites because of the presence of a
hydroxyl group in the phenolic structure (Helander et al.,
1998; Ultee et al., 2002; Calsamiglia et al., 2007).
Furthermore, the small molecular weight of thymol allows
it to gain access to the cell membrane through the pores of
the external wall (Calsamiglia et al., 2007). The strong
and widespectrum activity against gram-positive and
gram-negative bacteria, the narrow margin of security
between an optimal and a toxic dose and the effects
reported, which were not always in the desired direction
(Castillejos et al., 2006; Calsamiglia et al., 2007), suggest
that its antimicrobial activity may be too strong and
nonspecific to modulate the fermentation in a complex
microbial environment such as the rumen (Calsamiglia
et al., 2007). he cumulative volume of gas production
20
10
0
0
20
40
60
80
Incubation times, (h)
100
120
Fig. 1: In vitro gas production volume of soybean meal and
Zataria multiflora extract (0.15/30 mL buffered rumen
fluid) was different incubation time
Table 1: Chemical composition of soybean meal (%)
Component
Dry Matter (DM)
Crude Protein (CP)
Non-Fiber Carbohydrate (NFC)
%
94.07
40.00
29.36
Gas production volumes (mL/200 mg DM) and
estimated parameters at differents incubation times of
soybean meal and Zataria multiflora extract (0.15 mL/30
mL buffered rumen fluid) are presented in Table 2 and 3.
The gas volumes for soybean meal in different
incubation times were higher than that of Zataria
multiflora extract (0.15 mL/30 mL buffered rumen fluid).
Gas volume at 12 h incubation (for 200 mg dry samples),
of Soybean Meal (SBM) and Zataria multiflora extract
(0.15 mL/30 mL buffered rumen fluid) were 46.24 and
24.34 mL/200 mg DM, respectively. Gas volume at 24 h
incubation (for 200 mg dry samples), of Soybean Meal
(SBM) and Zataria multiflora extract (0.15 mL/30 mL
buffered rumen fluid) were 56.38 and 36.41 (mL/200 mg
DM), respectively. Gas volume at 48 h incubation (for
200 mg dry samples), of Soybean Meal (SBM) and
Zataria multiflora extract (0.15 mL/30 mL buffered
rumen fluid) were 62.12 and 38.01 (mL/200 mg DM),
respectively. Gas volume at 72 h incubation (for 200 mg
dry samples), of soybean meal and Zataria multiflora
extract (0.15 mL/30 mL buffered rumen fluid) were 62.91
and 41.62, respectively. Gas volume at 96 h incubation
(for 200 mg dry samples), of soybean meal and Zataria
multiflora extract (0.15 mL/30 mL buffered rumen fluid)
were 63.72 and 42.82, respectively. the gas production
from soluble fraction (a), the gas production from
insoluble fraction (b), rate constant of gas production
during incubation (c) and the potential gas production
(a+b) contents of soybean meal (control) were 4.42
(mL/200 mg DM), 67.1 (mL/200 mg DM), 0.113 (mL/h)
and 71.52 (mL/200 mg DM), the gas production from
soluble fraction (a), the gas production from insoluble
fraction (b), rate constant of gas production during
incubation (c) and the potential gas production (a+b)
352
Curr. Res. J. Biol. Sci., 4(4): 350-354, 2012
Table 2:In vitro gas production volumes (mL/200 mg DM) of soybean meal and Zataria multiflora extract (0.15 mL/30 mL buffered rumen fluid)
at different incubation times
Incubation times
--------------------------------------------------------------------------------------------------------------------------------------------------------Treatment
2
4
6
8
12
24
48
72
96
No Additive
9.31
19.96
27.623
7.26
46.24
56.38
62.12
62.91
63.72
ZME 0.15
4.92
7.47
9.04
14.11
24.34
36.41
38.01
41.62
42.82
P Value
0.0001
p<0.001
p<0.001
p<0.001
0.0001
0.0007
0.0004
0.0007
0.0012
SEM
2.428
6.868
10.194
12.723
12.093
18.186
13.428
11.958
11.774
Table 3: The estimated parameters from the gas production were
soybean meal and Zataria multiflora extract (0.15 mL/30 mL
buffered rumen fluid)
Estimated parameters
---------------------------------------------------------------Treatment
a
b
|a|+b
c
No additive
4.42
67.1
71.52
0.113
ZME 0.15
3.52
45.54
49.06
0.07
P-value
0.0476
0.0012
0.0014
p<0.001
SEM
0.605
12.569
12.693
0.023
a+b :
SBM :
DM :
ZME :
The potential gas production (mL/200 mg DM)
Soybean meal
Dry matter
Zataria multiflora extract
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ABBREVIATIONS
GP : Gas production technique
NFC : Non-Fibrous Carbohydrate
(a) : The gas production from soluble fraction (mL/200
mg DM)
b
: The gas production from insoluble fraction
(mL/200 mg DM)
c
: Rate constant of gas production during incubation
(mL/h)
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