Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol83.Issue2.Feb2000:
Animal Feed Science and Technology
83 (2000) 127±138
In vitro study of the rumen and hindgut fermentation
of ®brous materials (meadow hay, beech sawdust,
wheat straw) in sheep
Zora VaÂradyovaÂ*, Imrich ZelenÏaÂk, Peter Siroka
Institute of Animal Physiology, Slovak Academy of Sciences, Kosice, Slovak Republic
Received 24 July 1998; received in revised form 19 March 1999; accepted 12 October 1999
Abstract
The in¯uence of both rumen and hindgut inocula of sheep on fermentation of ®brous materials in
vitro has been investigated. Different ®brous materials (meadow hay, beech sawdust, wheat straw)
and cellulose were used as substrates. The study was carried out to compare: (1) fermentation of
substrates with rumen and hindgut inocula, (2) fermentation of meadow hay (reference substrate)
and other substrates, (3) fermentation of the two types of cellulose (amorphous and crystalline), and
(4) fermentation of treated ®brous materials (treated beech sawdust by de®bration and impregnation
and fungal treated wheat straw) and untreated ®brous materials. Hindgut fermentation of ®brous
materials was associated with decreased dry matter and neutral detergent ®bre degradabilities, and
also methane and total gas production. The calculated hydrogen recoveries with hindgut inoculum
showed a tendency to lower values as compared to the rumen inoculum. Signi®cant differences
were found between meadow hay and other ®brous materials, between both celluloses and between
treated and untreated ®brous materials. The positive correlation between hydrogen recoveries and
methane production of untreated wheat straw with a hindgut inoculum suggested the presence of
reductive acetogenesis with the hindgut inoculum. It can be concluded that reductive acetogenesis
with a hindgut inoculum instead of methanogenesis may increase the energetic yield from VFA per
substrate, and to some extent also the energetic yield for the host animal. # 2000 Elsevier Science
B.V. All rights reserved.
Keywords: Rumen; Hindgut; Fermentation; In vitro; Degradabilities
*
Corresponding author. Tel.: 42-1-9576-3121/42-1-9563-36268; fax: 42-1-9576-2162.
E-mail address: varady@saske.sk (Z. VaÂradyovaÂ).
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 1 2 1 - 2
128
Z. VaÂradyova et al. / Animal Feed Science and Technology 83 (2000) 127±138
1. Introduction
It is well known that the ability to convert ®brous materials to quality protein for
human nutrition is the advantage of ruminants. The plant cell wall polysaccharides may
be utilized in ruminant metabolism after their fermentation by the microbial population
found in the digestive tract. In spite of similarity in rumen and hindgut fermentation there
exist important differences: these include lower production of methane and hydrogen
recoveries for the hindgut, as well as absence of ciliate protozoa and the presence of
reductive acetogenesis or dissimilatory sulfate reduction (Demeyer and De Graeve, 1991;
Immig et al., 1996). The presence of high mucin and free amino acids concentration in
the hindgut, in contrast to the rumen, may be a factor responsible for the induction of
reductive acetogenesis in the hindgut (Demeyer et al., 1996). In ruminants microbial
digestion in the hindgut occurs to a lesser degree than in the rumen except for pelleted
diet and forages with a very high content of lignin (Hoover, 1978). The extent of
microbial digestion in the hindgut remains extremely variable and depends not only
upon species and nature of the diet, where the proportion of ligni®ed products rich in
plant cell walls mainly determines the development of the microbial biomass, but also on
the form of presentation particularly the size of the fodder particles (Tisserand, 1989).
The measurement of fermentation parameters by the modi®ed pressure transducer
technique (Theodorou et al., 1994) in vitro, were in the present study used for both rumen
and hindgut inocula. Gas measurements provide useful data on the digestion kinetics of
both soluble and insoluble fractions of feedstuffs (Getachew et al., 1998). The objectives
of the present in vitro study were as follows: (1) to compare the in¯uence of rumen and
hindgut inocula on the fermentation of ®brous substrates (meadow hay, beech sawdust
and wheat straw), (2) to compare the fermentation of meadow hay and other ®brous
substrates, (3) to compare the two types of cellulose (amorphous and crystalline), and (4)
to compare the differences between the fermentation of treated materials (treated beech
sawdust by de®bration and impregnation and fungal treated wheat straw) and untreated
materials.
2. Material and methods
2.1. Inocula, method of incubation and substrates
The ruminal and hindgut inocula used in the present experiment were obtained from
two slaughtered sheep. Samples were transferred to the laboratory, squeezed through four
layers of gauze and purged with CO2. The inocula were mixed with Mc Dougall's buffer
(Mc Dougall, 1948) at a ratio of 1 : 2 and 35 ml doses (®ve replicates each for rumen and
hindgut inocula) were dispensed by an automatic pump into preheated 120 ml serum
bottles containing 0.25 g of substrate and incubated in the incubator for 72 h at
39 0.58C. Five replicate bottles were also used for the controls (rumen or hindgut
inoculum, no substrate). The following seven substrates were used: cellulose amorphous
(CA), cellulose crystalline (CC), meadow hay (MH), treated beech sawdust (TS),
untreated beech sawdust (US), treated wheat straw (TWS) and untreated wheat straw
Z. VaÂradyova et al. / Animal Feed Science and Technology 83 (2000) 127±138
129
(UWS). Meadow hay, TS, TWS and UWS had a particle size of 0.15±0.4 mm. Treatment
of beech sawdust and fungal treatment of wheat straw (Phelinus laevigatus 657I) was
described by ZelenÏaÂk et al., 1987; JalcÏ et al., 1997.
2.2. Gas measurements
The volume of released accumulated gas was measured after 72 h by the
pressure transducer technique (VaÂradyova et al., 1998). The metering system consisted
of a three-way valve, mechanical pressure manometer, gas-tight-syringe and needle.
The three-way valve was connected with the pressure Ð manometer (to measure the
pressure in the serum bottles) and gas-tight syringe (to measure the volume of gas
production). The third port was connected with a needle by a hose. The needle was
used to punch the rubber stopper on the serum bottle. Gases from each fermentation
bottle were collected in a 2 ml glass syringe (for each bottle separately) at the end of
the incubations and immediately analysed for methane concentration by gas
chromatography.
2.3. VFA analysis
The concentrations of volatile fatty acids (VFA) in the medium at 72 h were
determined by gas chromatography (Cottyn and Boucque, 1968) using crotonic acid as
the internal standard and the Perkin±Elmer 8500 gas chromatograph.
2.4. Estimation of DM and NDF degradability
Dry matter degradability (DMD) was estimated from the difference of substrate weight
before and after incubation. The contents of the bottles were transferred into a tube and
centrifuged at 3500 g for 10 min. The residues were washed twice with distilled water,
centrifuged and dried to constant weight at 105 8C (Mellenberger et al., 1970). Samples
(1 0.001) g were analysed for neutral detergent ®bre (NDF) degradability by the
method of Van Soest et al. (1991).
2.5. Calculations and statistical analysis
Hydrogen recoveries were calculated according to Demeyer and Van Nevel (1975);
Marounek et al. (1997). The following equations were used:
Recovery 2H accepted/2H released
2H accepted 4M 2P 2B 4V
2H released 2H P 4B 3V
where M is methane, A is acetate, P is propionate, B is butyrate and V is valerate.
The means of individual parameters were compared using the Student±Newman±Keuls
test (GraphPad InStat, GraphPad Software, Inc. San Diego, USA).
130
Z. VaÂradyova et al. / Animal Feed Science and Technology 83 (2000) 127±138
3. Results
3.1. Rumen and hindgut fermentation of ®brous materials (MH, TS, US, TWS, UWS)
As compared to rumen inocula (Table 1, Fig. 1) signi®cantly lower values (P < 0.001)
were obtained in DM and NDF degradability of MH, TS and TWS with hindgut inocula.
The DM degradability was lower (P < 0.001) also for UWS. Total gas and methane
production of ®brous materials (MH, TS, TWS, UWS) were decreased with hindgut
inocula (Figs. 2 and 3). The hydrogen recovery of these ®brous materials was lower with
hindgut inocula, but signi®cantly different values (P < 0.01) were obtained only for UWS
(Fig. 4). The total VFA production of TS was signi®cantly lower with hindgut inocula
compared to rumen inocula (Table 2, Fig. 5). The mol% for propionate of UWS was
lower (P < 0.001) with hindgut inocula. The values of mol% for butyrate of UWS and
MH were signi®cantly higher (P < 0.001; P < 0.01) with hindgut inocula. The value of
mol% for iso-valerate of MH was lower (P < 0.001) with hindgut inocula (Table 3). The
mol% for iso-butyrate of TWS was higher (P < 0.001) with hindgut inocula. As compared
to rumen inocula the values of mol% for iso-butyrate and valerate of UWS were
signi®cantly higher with hindgut inocula.
3.2. Meadow hay (MH) compared to other ®brous substrates (TS, US, TWS, UWS)
Result are presented in Tables 1±3. Compared to values for MH, NDF degradability
was higher for TWS for rumen inocula and for TS and TWS for hindgut inocula, whereas
it was lower for US and UWS for both inocula. In contrast, where measurable, total gas,
methane gas content and total VFA production, and concentrations of acetic acid for TS,
US, TWS and UWS were signi®cantly lower than for MH for both inocula, as was butyric
Fig. 1. The DM degradability of cellulose amorphous (CA), cellulose crystaline (CC), meadow hay (MH),
treated beech sawdust (TS), untreated beech sawdust (US), treated wheat straw (TWS) and untreated wheat
straw (UWS) incubated with rumen or hindgut inocula in vitro, for 72 h.
Rumen degradability
DM (%)
NDF (%)
Total gas (ml/bottle)
Methane (10ÿ2 ml/ml)
2H Rec (%)
Hindgut degradability
DM (%)
NDF (%)
Total gas (ml/bottle)
Methane (10ÿ2 ml/ml)
2H Rec (%)
a
CA
CC
MH
TS
US
TWS
UWS
86.0 2.1
±
59.5 1.3
4.1 0.1
67.0 2.7
81.2 4.0
±
51.5 3.3
3.5 0.1
75.2 2.8
55.8 2.5
71.7 0.6
30.9 0.8
3.6 0.9
32.0 2.9
59.9 4.8
71.1 0.1
26.3 1.6b
2.1 0.1b
52.0 4.1b
5.1 1.9b,g
7.8 0.5b,g
1.8 0.9b,g
0.5 0.1b,g
d
57.6 1.6
78.4 0.4b
22.7 0.8b
2.9 0.1
50.6 4.4b
37.5 2.3b,g
40.1 0.1b,g
19.9 2.2b,b
3.0 0.2
61.4 4.5b
88.9 1.5
±
55.0 0.8***
2.5 0.2***
69.3 1.9
80.0 4.0
±
53.0 1.9
2.4 0.2**
66.0 1.7
44.6 4.1***
61.4 0.1***
26.4 1.2***
2.9 0.2*
33.6 1.1
41.1 6.8***
66.4 0.1***,b
23.0 0.8**,a
1.6 0.2b
48.2 0.7b
5.1 0.6b,g
8.2 0.1b,g
d
d
d
40.9 2.3***
65.6 0.1***,b
18.2 0.6***,b
1.9 0.2**,a
47.6 2.6b
18.8 1.9***,b,g
40.2 0.1b,g
9.1 0.2***,b,g
1.2 0.4***,b
44.7 3.5**,b
P
83 (2000) 127±138
In vitro study of the rumen and hindgut fermentation
of ®brous materials (meadow hay, beech sawdust,
wheat straw) in sheep
Zora VaÂradyovaÂ*, Imrich ZelenÏaÂk, Peter Siroka
Institute of Animal Physiology, Slovak Academy of Sciences, Kosice, Slovak Republic
Received 24 July 1998; received in revised form 19 March 1999; accepted 12 October 1999
Abstract
The in¯uence of both rumen and hindgut inocula of sheep on fermentation of ®brous materials in
vitro has been investigated. Different ®brous materials (meadow hay, beech sawdust, wheat straw)
and cellulose were used as substrates. The study was carried out to compare: (1) fermentation of
substrates with rumen and hindgut inocula, (2) fermentation of meadow hay (reference substrate)
and other substrates, (3) fermentation of the two types of cellulose (amorphous and crystalline), and
(4) fermentation of treated ®brous materials (treated beech sawdust by de®bration and impregnation
and fungal treated wheat straw) and untreated ®brous materials. Hindgut fermentation of ®brous
materials was associated with decreased dry matter and neutral detergent ®bre degradabilities, and
also methane and total gas production. The calculated hydrogen recoveries with hindgut inoculum
showed a tendency to lower values as compared to the rumen inoculum. Signi®cant differences
were found between meadow hay and other ®brous materials, between both celluloses and between
treated and untreated ®brous materials. The positive correlation between hydrogen recoveries and
methane production of untreated wheat straw with a hindgut inoculum suggested the presence of
reductive acetogenesis with the hindgut inoculum. It can be concluded that reductive acetogenesis
with a hindgut inoculum instead of methanogenesis may increase the energetic yield from VFA per
substrate, and to some extent also the energetic yield for the host animal. # 2000 Elsevier Science
B.V. All rights reserved.
Keywords: Rumen; Hindgut; Fermentation; In vitro; Degradabilities
*
Corresponding author. Tel.: 42-1-9576-3121/42-1-9563-36268; fax: 42-1-9576-2162.
E-mail address: varady@saske.sk (Z. VaÂradyovaÂ).
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 1 2 1 - 2
128
Z. VaÂradyova et al. / Animal Feed Science and Technology 83 (2000) 127±138
1. Introduction
It is well known that the ability to convert ®brous materials to quality protein for
human nutrition is the advantage of ruminants. The plant cell wall polysaccharides may
be utilized in ruminant metabolism after their fermentation by the microbial population
found in the digestive tract. In spite of similarity in rumen and hindgut fermentation there
exist important differences: these include lower production of methane and hydrogen
recoveries for the hindgut, as well as absence of ciliate protozoa and the presence of
reductive acetogenesis or dissimilatory sulfate reduction (Demeyer and De Graeve, 1991;
Immig et al., 1996). The presence of high mucin and free amino acids concentration in
the hindgut, in contrast to the rumen, may be a factor responsible for the induction of
reductive acetogenesis in the hindgut (Demeyer et al., 1996). In ruminants microbial
digestion in the hindgut occurs to a lesser degree than in the rumen except for pelleted
diet and forages with a very high content of lignin (Hoover, 1978). The extent of
microbial digestion in the hindgut remains extremely variable and depends not only
upon species and nature of the diet, where the proportion of ligni®ed products rich in
plant cell walls mainly determines the development of the microbial biomass, but also on
the form of presentation particularly the size of the fodder particles (Tisserand, 1989).
The measurement of fermentation parameters by the modi®ed pressure transducer
technique (Theodorou et al., 1994) in vitro, were in the present study used for both rumen
and hindgut inocula. Gas measurements provide useful data on the digestion kinetics of
both soluble and insoluble fractions of feedstuffs (Getachew et al., 1998). The objectives
of the present in vitro study were as follows: (1) to compare the in¯uence of rumen and
hindgut inocula on the fermentation of ®brous substrates (meadow hay, beech sawdust
and wheat straw), (2) to compare the fermentation of meadow hay and other ®brous
substrates, (3) to compare the two types of cellulose (amorphous and crystalline), and (4)
to compare the differences between the fermentation of treated materials (treated beech
sawdust by de®bration and impregnation and fungal treated wheat straw) and untreated
materials.
2. Material and methods
2.1. Inocula, method of incubation and substrates
The ruminal and hindgut inocula used in the present experiment were obtained from
two slaughtered sheep. Samples were transferred to the laboratory, squeezed through four
layers of gauze and purged with CO2. The inocula were mixed with Mc Dougall's buffer
(Mc Dougall, 1948) at a ratio of 1 : 2 and 35 ml doses (®ve replicates each for rumen and
hindgut inocula) were dispensed by an automatic pump into preheated 120 ml serum
bottles containing 0.25 g of substrate and incubated in the incubator for 72 h at
39 0.58C. Five replicate bottles were also used for the controls (rumen or hindgut
inoculum, no substrate). The following seven substrates were used: cellulose amorphous
(CA), cellulose crystalline (CC), meadow hay (MH), treated beech sawdust (TS),
untreated beech sawdust (US), treated wheat straw (TWS) and untreated wheat straw
Z. VaÂradyova et al. / Animal Feed Science and Technology 83 (2000) 127±138
129
(UWS). Meadow hay, TS, TWS and UWS had a particle size of 0.15±0.4 mm. Treatment
of beech sawdust and fungal treatment of wheat straw (Phelinus laevigatus 657I) was
described by ZelenÏaÂk et al., 1987; JalcÏ et al., 1997.
2.2. Gas measurements
The volume of released accumulated gas was measured after 72 h by the
pressure transducer technique (VaÂradyova et al., 1998). The metering system consisted
of a three-way valve, mechanical pressure manometer, gas-tight-syringe and needle.
The three-way valve was connected with the pressure Ð manometer (to measure the
pressure in the serum bottles) and gas-tight syringe (to measure the volume of gas
production). The third port was connected with a needle by a hose. The needle was
used to punch the rubber stopper on the serum bottle. Gases from each fermentation
bottle were collected in a 2 ml glass syringe (for each bottle separately) at the end of
the incubations and immediately analysed for methane concentration by gas
chromatography.
2.3. VFA analysis
The concentrations of volatile fatty acids (VFA) in the medium at 72 h were
determined by gas chromatography (Cottyn and Boucque, 1968) using crotonic acid as
the internal standard and the Perkin±Elmer 8500 gas chromatograph.
2.4. Estimation of DM and NDF degradability
Dry matter degradability (DMD) was estimated from the difference of substrate weight
before and after incubation. The contents of the bottles were transferred into a tube and
centrifuged at 3500 g for 10 min. The residues were washed twice with distilled water,
centrifuged and dried to constant weight at 105 8C (Mellenberger et al., 1970). Samples
(1 0.001) g were analysed for neutral detergent ®bre (NDF) degradability by the
method of Van Soest et al. (1991).
2.5. Calculations and statistical analysis
Hydrogen recoveries were calculated according to Demeyer and Van Nevel (1975);
Marounek et al. (1997). The following equations were used:
Recovery 2H accepted/2H released
2H accepted 4M 2P 2B 4V
2H released 2H P 4B 3V
where M is methane, A is acetate, P is propionate, B is butyrate and V is valerate.
The means of individual parameters were compared using the Student±Newman±Keuls
test (GraphPad InStat, GraphPad Software, Inc. San Diego, USA).
130
Z. VaÂradyova et al. / Animal Feed Science and Technology 83 (2000) 127±138
3. Results
3.1. Rumen and hindgut fermentation of ®brous materials (MH, TS, US, TWS, UWS)
As compared to rumen inocula (Table 1, Fig. 1) signi®cantly lower values (P < 0.001)
were obtained in DM and NDF degradability of MH, TS and TWS with hindgut inocula.
The DM degradability was lower (P < 0.001) also for UWS. Total gas and methane
production of ®brous materials (MH, TS, TWS, UWS) were decreased with hindgut
inocula (Figs. 2 and 3). The hydrogen recovery of these ®brous materials was lower with
hindgut inocula, but signi®cantly different values (P < 0.01) were obtained only for UWS
(Fig. 4). The total VFA production of TS was signi®cantly lower with hindgut inocula
compared to rumen inocula (Table 2, Fig. 5). The mol% for propionate of UWS was
lower (P < 0.001) with hindgut inocula. The values of mol% for butyrate of UWS and
MH were signi®cantly higher (P < 0.001; P < 0.01) with hindgut inocula. The value of
mol% for iso-valerate of MH was lower (P < 0.001) with hindgut inocula (Table 3). The
mol% for iso-butyrate of TWS was higher (P < 0.001) with hindgut inocula. As compared
to rumen inocula the values of mol% for iso-butyrate and valerate of UWS were
signi®cantly higher with hindgut inocula.
3.2. Meadow hay (MH) compared to other ®brous substrates (TS, US, TWS, UWS)
Result are presented in Tables 1±3. Compared to values for MH, NDF degradability
was higher for TWS for rumen inocula and for TS and TWS for hindgut inocula, whereas
it was lower for US and UWS for both inocula. In contrast, where measurable, total gas,
methane gas content and total VFA production, and concentrations of acetic acid for TS,
US, TWS and UWS were signi®cantly lower than for MH for both inocula, as was butyric
Fig. 1. The DM degradability of cellulose amorphous (CA), cellulose crystaline (CC), meadow hay (MH),
treated beech sawdust (TS), untreated beech sawdust (US), treated wheat straw (TWS) and untreated wheat
straw (UWS) incubated with rumen or hindgut inocula in vitro, for 72 h.
Rumen degradability
DM (%)
NDF (%)
Total gas (ml/bottle)
Methane (10ÿ2 ml/ml)
2H Rec (%)
Hindgut degradability
DM (%)
NDF (%)
Total gas (ml/bottle)
Methane (10ÿ2 ml/ml)
2H Rec (%)
a
CA
CC
MH
TS
US
TWS
UWS
86.0 2.1
±
59.5 1.3
4.1 0.1
67.0 2.7
81.2 4.0
±
51.5 3.3
3.5 0.1
75.2 2.8
55.8 2.5
71.7 0.6
30.9 0.8
3.6 0.9
32.0 2.9
59.9 4.8
71.1 0.1
26.3 1.6b
2.1 0.1b
52.0 4.1b
5.1 1.9b,g
7.8 0.5b,g
1.8 0.9b,g
0.5 0.1b,g
d
57.6 1.6
78.4 0.4b
22.7 0.8b
2.9 0.1
50.6 4.4b
37.5 2.3b,g
40.1 0.1b,g
19.9 2.2b,b
3.0 0.2
61.4 4.5b
88.9 1.5
±
55.0 0.8***
2.5 0.2***
69.3 1.9
80.0 4.0
±
53.0 1.9
2.4 0.2**
66.0 1.7
44.6 4.1***
61.4 0.1***
26.4 1.2***
2.9 0.2*
33.6 1.1
41.1 6.8***
66.4 0.1***,b
23.0 0.8**,a
1.6 0.2b
48.2 0.7b
5.1 0.6b,g
8.2 0.1b,g
d
d
d
40.9 2.3***
65.6 0.1***,b
18.2 0.6***,b
1.9 0.2**,a
47.6 2.6b
18.8 1.9***,b,g
40.2 0.1b,g
9.1 0.2***,b,g
1.2 0.4***,b
44.7 3.5**,b
P