Methane Production Potential of Feed Ingredients As Measured by in Vitro Gas Test
Methane Production Potential of Feed Ingredients As Measured by in Vitro Gas Test
Methane Production Potential of Feed Ingredients As Measured by in Vitro Gas Test
ABSTRACT : This study was conducted to investigate in vitro methane production of feed ingredients and relationship between the
content of crude nutrients and methane production. Feed ingredients (total 26) were grouped as grains (5 ingredients), brans and hulls
(8), oil seed meals (9) roughages (3), and animal by-product (1) from their nutrient composition and their methane production protential
were measured by in vitro gas test. Among the groups, the in vitro methane productions for both 6 and 24 h incubation were highest in
grains, followed by brans and hulls, oil meals and roughages, animal byproducts. Within the group of grains, methane production from
wheat flour was the highest, followed by wheat, corn, tapioca, and then oat. Within the brans and hulls, soybean hull showed the highest
methane production and cotton seed hull, the lowest. Methane production from oil meals was lower compared with grains and brans and
hulls, and in decreasing order production from canola meal was followed by soybean meal, coconut meal, and corn germ meal (p<0.01).
Three ingredients were selected and the interactions among feed ingredients were evaluated for methane production. Correlation
coefficient between measured and estimated values of the combinations were 0.91. Methane production from each feed ingredient was
decreased with increasing amount of crude fiber (CF), protein (CP) and ether extract (EE), whereas positive relationship was noted with
the concentrations of N-free extract (NFE). The multiple regression equation (n=134) for methane production and nutrient
concentrations was as follows. Methane production (ml/0.2 g DM)=(0.032×CP)-(0.057×EE)-(0.012×CF)+(0.124×NFE) (p<0.01;
R2=0.929). Positive relationship was noted for CP and NFE and negative relationship for CF and EE. It seems possible to predict
methane production potential from nutritional composition of the ingredients for their effective application on formulating less methane
emitting rations. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 8 : 1143-1150)
producing formulation in ruminant diets. production was measured to investigate the interaction of
substrate utilization for methane production by rumen
MATERIALS AND METHODS microbes among feed ingredients. Three feed ingredients
were selected in each group, grain, bran and oil seed meal
Animals and combined to give 27 different combinations. Methane
Ruminally cannulated, Korean native cattle, Hawoo productions of combinated feed ingredients were compared
steer (Body weight: 500 kg) were fed with diet containing with the expected.
80% concentrate and 20% roughage (7.2 Kg of DM/d). Gas test : In vitro methane production was conducted by
Feed was provided twice a day and water and mineral block Menke's gas test (Menke, 1979). A total of 200 mg (DM;
were offered ad libitum. dry matter basis) of feed ingredients were loaded in glass
syringe (volume: 100 ml) and treated with grease not to
In vitro methane production loose fluid and gas produced during incubation. Rumen
Preparation of feed ingredients : Total 26 feed fluid were obtained from Hanwoo just before feeding. The
ingredients namely 5 grains, 8 brans and hulls group, 9 oil pH were determined immediately. Feed particles were
seed meals, 3 roughages and 1 animal byproduct were removed by squeezing it through eight layers of cheesecloth.
tested. All feed ingredients were milled with Wiley Mill Feed ingredients were incubated triplicate with rumen fluid
and screened with 1 mm mesh sieve. Moisture, crude taken from two steers.
protein, crude fiber and ether extract were analysed The composition of incubation medium was 400 ml
according to A.O.A.C. (1990) method and shown in Table 1. distilled water, trace element solution, buffer solution, main
Combination of feed ingredients : Equal amount of feed element solution as was in Menke’s method (Menke, 1979).
ingredients were combinated to give a 200 mg and methane The medium were saturated CO2 gas and pH was adjusted
METHANE PRODUCTION OF FEED INGREDIENTS 1145
8
which has same nutritional composition except for fat. Thus,
7 high content of fat (unsaturated fatty acids) in rice bran
(ml/0.2g DM)
PRD(ml/0.2 g DM)
Oil seed Brans and B/A 10
Grains 6 h 24 h (A) (B)
Meals* Hulls**
Corn SBM WB 4.92 10.54 9.08 0.86 9
Corn SBM SBH 3.49 9.88 10.08 1.02
Corn SBM RB 4.00 7.86 7.87 1.00 8
Corn RSM WB 4.45 8.68 8.49 0.98
Corn RSM SBH 3.92 9.85 9.47 0.96 7
Corn RSM RB 3.94 7.02 7.32 1.04
6 y=0.9597x+0.1 (R2=0.8099)
Corn CSM WB 4.42 8.02 8.12 1.01
Corn CSM SBH 3.57 9.16 9.12 1.00
5
Corn CSM RB 3.86 6.65 6.92 1.04 5 6 7 8 9 10 11
Wheat SBM WB 4.76 9.23 9.46 1.02
Wheat SBM SBH 4.56 10.63 10.46 0.98
Obeserved methane production (ml/0.2 g DM)
Wheat SBM RB 5.00 9.02 8.25 0.91
Figure 2. Relationship between the measured and the predicted
Wheat RSM WB 5.24 9.28 8.86 0.95
value for 24 h in vitro methane production of feed ingredient
Wheat RSM SBH 4.11 9.21 9.84 1.07
combination.
Wheat RSM RB 4.61 7.97 7.69 0.96
Wheat CSM WB 5.31 9.11 8.50 0.93
possible without consideration of feed ingredient interaction.
Wheat CSM SBH 4.31 10.0 9.50 0.95
Wheat CSM RB 4.67 8.15 7.30 0.90
Oat SBM WB 4.71 8.32 7.85 0.94 Relation between methane production and nutritional
Oat SBM SBH 3.74 9.90 8.85 0.89 constituents
Oat SBM RB 4.72 7.87 7.42 0.94 Crude fiber : The relationship between the content of
Oat RSM WB 4.85 8.32 7.29 0.88 crude fiber and methane production in feed ingredients
Oat RSM SBH 4.09 8.98 8.28 0.92 investigated are shown in Table 4. Methane productions
Oat RSM RB 4.49 7.244 6.14 0.85 decreased as the content of crude fiber increased. Several
Oat CSM WB 4.59 7.47 6.90 0.92
attempts have been made to predict methane production by
Oat CSM SBH 3.54 8.21 7.91 0.96
determining the amount of crude nutrients in cattle and
Oat CSM RB 4.18 6.42 5.73 0.89
SEM 0.483 1.07 1.15 - sheep (Holter and Young, 1992; Shibata et al., 1994) and it
* SBM: Soybean meal, CSM: Cottonseed meal, RSM: Rapeseed meal, is known that crude fiber is an important component in
** WB: Wheat bran, SBH: Soy bean hull, RB: Rice bran methane production. Miller (1995) reported that feed
*** PRD: Predicted value, Mathematical average of individual in vitro ingredients rich in crude fiber stimulated some species of
methane production for 24 h.
microorganism within the cellulolytic-methanogen
production : To investigate interactions among feed consortium which serve to couple the degradation of
ingredients for methane production, three ingredients were carbohydrates with the use of H2 for the reduction of CO2 to
selected from each group based on their level of methane methane.
which showed significant differences. Methane production Feed ingredients were grouped according to their
from the combinations was measured and compared with compositional characteristics as grains, brans and hulls, oil
their predicted value (PRD, mathematical average of three seed meals and regression for methane production due to
feed ingredients) estimated from individual production crude fiber for each group was developed. Thus, equations
(Table 3). in Table 4 indicate that methane production decreases as
There were no differences for methane production crude fiber increases in grains. It was less affected by crude
between actual and predicted values. The methane fiber in brans and hulls. Oil seed meals seemed to be less
production values were higher in soybean meal, wheat, related to crude fiber than brans and hulls and and grain. In
soybean hull combinations and lower in cotton seed meal, vitro methane production of feed ingredients with more
oat, rice which showed less methane production (p<0.01). crude fiber were negatively related with the amount of
Standard deviations between practical and predicted values crude fiber.
were less than 5% and R2 was 0.8099 (Figure 2). There Grains usually contain hard out layer, hull, which is cell
seemed to be no substrate interaction for methane wall component hardly degraded by rumen microbes and
production among feed ingredients and the estimation of this might cause negative relationship with methane
methane production from a feed ingredients would be production.
Crude protein : The relationship of methane production
1148 LEE ET AL.
Table 4. Regression analysis of methane production by each crude constituent: total, grain, brans and hulls, oil seed meals
Feed ingredients n1) Regression equation R2
Crude fiber Total 134 y=0.0011x2-0.1123x+7.839 0.076
Grains 25 y=-0.7014x2-0.1638x+11.618 0.694
brans and hulls 33 y=-0.0047x2+0.3362x+3.6224 0.471
Oil seed meals 38 y=0.0194x2-0.5976x+9.6753 0.145
Crude protein Total 134 y=-0.0021x2+0.0716x+6.7667 0.182
Grains 25 y=-0.0368x2+0.5469x+8.9763 0.035
brans and hulls 33 y=0.0463x2-1.7193x+22.816 0.364
Oil seed meals 48 y=-0.00072x2+0.0276x+5.8952 0.041
NFE Total 134 y=0.1098x+1.1048 0.515
Grains 25 y=0.2317x-8.3677 0.673
brans and hulls 33 y=0.0169x+6.8142 0.008
Oil seed meals 48 y=0.0545x+3.6945 0.210
Ether extract Total 134 y=-0.0006x2-0.172x+7.2532 0.071
Grains 25 y=-0.4259x+10.732 0.158
brans and hulls 33 y=-1.4839x2+5.871x+4.3041 0.182
Oil seed meals 48 y=-0.1794x2+1.743x+3.3747 0.481
1) 2
n: number of samples, R : correlation coefficient.
Table 5. Estimation of methane production by crude constituent: total, grain, brans and hulls, oil seed meals
Significacne
Feed ingredients n1) Regression equation R2
of regression
Total 134 CH4=0.032CP-0.057CFat-0.012CF+0.124NFE 0.929 **
Grains 25 CH4=0.082CP-0.045CFat-0.017CF+0.129NFE 0.990 **
Brans and hulls 33 CH4=-0.065CP-0.041CFat+0.150CF+0.115NFE 0.988 **
Oil seed meals 48 CH4=0.062CP-0.067CFat-0.024CF+0.099NFE 0.962 **
Forage 15 CH4=0.430CP+0.105CF-0.078NFE 0.986 **
1)
n: number of samples, R2 : correlation coefficient, **: p<0.01.
and crude protein is shown in Table 4. The methane Ether extracts : Fat and other compounds included in
production was found to be decreased as the crude protein the ether extract fraction are mostly not fermented by rumen
was increased (R2: 0.182). Brans and hulls showed increase microbes, and especially unsaturated fatty acids are known
but grains and oil seed meals showed tendency to decrease to inhibit the methanogenic microbial system (Czerkowski
the methane production with the increase in the amount of et al. 1966; Demeyer and Van Nevel, 1975). Hydrogenation
crude protein. of unsaturated fatty acid increases propionate synthesis,
Getachew et al. (1998) reported that NH4, which was inhibits protozoa and cellulolytic bacterial activity and
released by protein degradation, combined with CO2, thereby affect the methane production (Czerkawski et al.,
methane substrate and resulted in less methane production. 1966). Also, Roger et al. (1992) reported that glycerol,
Kirchgessner et al. (1994) also reported that crude protein released from fat hydrolysis suppress the cellulolytic
produce relatively little methane. Kurihara et al. (1997) bacteria activity. Those trends were not clearly shown in
found decreased methane in cattle fed more than this results but as a whole, ether extract tends to reduce the
maintenance and also when the crude protein of diet was methane production.
increased. Generally the relationship between nutrient composition
Nitrogen free extract : The relationship of methane and methane production among feed ingredients was not
production and NFE is shown in Table 4. NFE containing clear. Correlation was high with crude fiber, NFE and low
high amount of easily fermentable carbohydrates such as with crude protein, ether extract in grains, showing that
starch, sugars, some cellulose which are highly soluble in crude fiber and NFE are important factors. In oil seed meals
rumen shift the fermentation pattern toward process which which is high in crude protein, correlation was high with
are linked to the consumption of H2 for propionate ether extracts. It seems that methane production of feed
consumption. ingredients are affected by nutritional composition and their
Shibata et al. (1992) from the multiple regression analyses interactions not by only single nutrient or a factor.
relating CH4 production to various nutrient intakes Estimation of methane production by nutritional
suggested that NFE had positive correlation with methane constituents : The results of multiple regression analyses
production. Our findings (Table 4) agrees with Shibata et al. relating methane production to various nutrient composition
(1992). Especially high correlation was estimated in grains. are summarized in Table 5.
METHANE PRODUCTION OF FEED INGREDIENTS 1149
Methane production in brans and hulls was positively In vitro gas measuring techniques for assessment of nutritional
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