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 Animal Feed Science and Technology
83 (2000) 127±138
*Corresponding author. Tel.:42-1-9576-3121/42-1-9563-36268; fax:42-1-9576-2162. E-mail address: varady@saske.sk (Z. VaÂradyovaÂ).
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 390.58C. 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
(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 3500gfor 10 min. The residues were washed twice with distilled water, centrifuged and dried to constant weight at 1058C (Mellenberger et al., 1970). Samples (10.001) 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:
Recovery2H accepted/2H released 2H accepted4M2P2B4V 2H released2HP4B3V
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).
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.
Table 1
Fermentation parameters (meansstandard deviation) of ®brous materials and celluloses with rumen or hindgut inocula after 72 h of incubation
CA CC MH TS US TWS UWS
bP<0.001 different from meadow hay; d indicates values
0.005. The percentage of methane has been stated in 1 ml of volume of total gas. 2H Rec, hydrogen recovery in rumen or hindgut inocula.
*P< 0.05. **P< 0.01.
***P< 0.001 differences between rumen and hindgut inocula b
P< 0.01. g
P< 0.001 differences between treated and untreated ®brous materials.
P< 0.05.
P< 0.001 differences between cellulose amorphous and cellulose crystalline.
acid mol% in hindgut inocula. Where acetic acid mol% were signi®cantly higher than for MH, propionic acid mol% were signi®cantly lower. Valeric acid mol% for TS and TWS was lower than MH for both inocula.
3.3. Comparison of the two cellulose types (CA and CC)
Comparison of the two types of cellulose (CA, CC) fermented with both rumen and hindgut inocula revealed signi®cant differences (Tables 1±3). Signi®cantly lower values Fig. 2. Total gas production 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.
Fig. 3. Methane gas production 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.
were obtained in total gas of CA and methane production of CA and CC with hindgut inocula as compared to rumen inocula. As compared to rumen inocula total VFA production and mol% for butyrate of CA were signi®cantly lower with hindgut inocula. The mol% values for iso-butyrate, iso-valerate and valerate of CC were signi®cantly higher with hindgut inocula as compared to rumen inocula. The DM degradability and Fig. 4. Hydrogen recovery 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.
Fig. 5. Total VFA production 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.
total VFA production of CC were signi®cantly lower as compared to CA with rumen and hindgut inocula. Total gas, mol% values for iso-butyrate and iso-valerate of CC were signi®cantly lower with rumen inocula as compared to CA. As compared to CA the mol% for iso-butyrate and iso-valerate of CC with hindgut inocula were signi®cantly higher. Table 2
VFA production (meanstandard deviation) during fermentation of different ®brous materials with rumen or hindgut inocula after 72 h of incubation
CA CC MH TS US TWS UWS
bP< 0.001 different from meadow hay; values0.005. *P< 0.05.
**P< 0.01.
***P< 0.001 differences between rumen and hindgut inocula. a
P< 0.05.
P< 0.05.
P< 0.001 differences between cellulose amorphous and cellulose crystalline.
Table 3
Production of iso-butyrate, iso-valerate, valerate of different ®brous materials with rumen or hindgut inocula after 72 h of incubation
Iso-butyrate (mol%) 0.7 1.0***,
0.5 0.4 d 0.6*** 0.3**
Iso-valerate (mol%) 0.6 0.7***,
0.3*** d d d d
Valerate (mol%) 0.6 0.7* 0.9 0.4a d 0.4a 0.7***
aP< 0.01.
bP< 0.001 different from meadow hay; d indicates values0.005. *P< 0.05.
**P< 0.01.
***P< 0.001 differences between rumen and hindgut inocula.
P< 0.05.
P< 0.01.
P< 0.001 differences between cellulose amorphous and cellulose crystalline.
3.4. Comparison of treated (TS, TWS) and untreated (US, UWS) ®brous materials
As compared to treated materials (TS, TWS) the DM and NDF degradability of untreated materials (US, UWS) were signi®cantly lower with both rumen and hindgut inocula (Table 1). Total gas and methane production of US was signi®cantly lower (P< 0.001) with rumen inocula as compared to TS. Total gas of UWS was signi®cantly lower with both inocula as compared to TWS. The values obtained for US indicated extremely low level (values < 0.005) of fermentation parameters (Tables 2 and 3). Total VFA production of UWS was signi®cantly lower with rumen inocula as compared to TWS (Table 2). The mol% for propionate was higher (P< 0.05) with rumen inocula as compared to TWS.
4. Discussion
In agreement with previous works (Hoover, 1978; Demeyer et al., 1989; Tisserand, 1989; Marounek et al., 1998) incubation with hindgut inoculum lead to lower digestibilities of DM and NDF of ®brous substrates as compared to rumen inocula. Not many data on fermentation parameters of hindgut fermentation of ®brous materials were found in literature as to enable comparison with our results. In our work hindgut inocula signi®cantly decreased total gas and methane production of ®brous materials. Lower production of methane in the hindgut as compared to the rumen was also reported in the experiments of Demeyer and De Graeve (1991); Marounek et al. (1998). In incubations with hindgut inoculum the obtained hydrogen recovery values were calculated in association with lower methanogenesis (Figs. 3 and 4). Similar results were obtained in other experiments (Marty and Demeyer, 1973; Demeyer and De Graeve, 1991; Marounek et al., 1998). A positive correlation between calculated hydrogen recoveries and methane production was observed in the rabbit hindgut (Piattoni et al., 1996; Marounek et al., 1997). Low values for hydrogen recoveries indicated the presence of reductive acetogenesis as a substantial source of acetate in the hindgut (Demeyer and De Graeve, 1991). However, inoculation of the rumen with the hindgut content did not induce reductive acetogenesis when evaluated by in vitro incubations (Immig et al., 1996). Most of the gastrointestinal degradability is known to occur in the rumen which is due to lack of cell wall degrading enzymes in the small intestine and the short retention time of ingesta in the hindgut as compared to the rumen (Rinne et al., 1997). The results revealed that degradability values of ®brous materials obtained from hindgut fermentation were lower in comparison to rumen fermentation, whereas the DM degradability of both celluloses was similar with both rumen and hindgut inocula. The differences between fermentation of hay and other substrates were similar with both inocula. DM degradability of hay and treated materials was similar, whereas total gas production and methane production values were different. Lower methane production and higher hydrogen recoveries of treated ®brous materials as compared to hay suggested better ef®ciency of treated beech sawdust and treated wheat straw. The cellulosic and ®brous materials in this study gave a low A : P ratio compared to MH. For MH the A : P ratio was 3.4±4.0 compared with TS and TWS with values of 1.5±1.8, which was due to
the relatively low propionate production of MH. Previously we tested the fermentation of barley straw, wheat straw, maize stalks, sugarcane bagasse and treated and untreated beech sawdust. The A : P ratios produced in the fermentation process ranged from 1.6 to 2.7 (ZelenÏaÂk et al., 1997), while MH fermented with the same inoculum yielded an A : P of 3.0 and more (ZelenÏaÂk, 1991). Similar results have been reported by Czerkawski (1986) who found an A : P ratio ranging from 3.0 to 4.5 for meadow hay fermented in the Rusitec. A : P ratios of 1.7 and 2.4 have been reported for cellulose and untreated wheat straw, respectively (Durand et al., 1988; Sunvold et al., 1995). The difference in A : P ratios may be related to either forage composition or the microbial population and metabolism, or both, and also the source of the inoculum may contribute to variation in A : P ratios (Marounek et al., 1985). The differences in total gas production observed in comparison with meadow hay and other substrates were caused by gas production associated with the rapidly digesting portion of the substrate. As the ®bre content of the forage increased, the amount of gas decreased (Scho®eld and Pell, 1995; Doane et al., 1997). Relationships between DM and NDF degradability and total gas production has also been recorded by other authors (BluÈmmel and érskov, 1993; Pell and Scho®eld, 1993; Doane et al., 1997).
Total gas and VFA production were in correlation with DM degradability (Figs. 1, 2 and 5). DM degradability of both celluloses (CA and CC) was higher than that of meadow hay for both inocula. However, the degradability of cellulose amorphous appeared to be always higher than that of cellulose crystalline. There is a correlation between the in vitro digestibility of various samples of cellulose and the crystallization index of these samples estimated by means of roentgenospectrometry. The higher the value of crystallization the lower is the degradability (Baker et al., 1975). Unlike the other substrates both celluloses examined in our work revealed similar DM degradability values for both inocula.
The results pointed at a high signi®cance of the treatment of ®brous materials as compared to untreated ones. The in¯uence of treatment of beech sawdust and fungal treatment of wheat straw on fermentation parameters in vitro has been reported (JalcÏ et al., 1997; ZelenÏaÂk et al., 1997). According to Baker et al., (1975) DM degradability of untreated oak and beech wood is 0±5%. In our previous work the DM degradability of untreated beech sawdust ranged from 0±10% (ZelenÏaÂk, 1991).
5. Conclusions
It was the objective of this study to compare fermentation of ®brous materials (meadow hay, beech sawdust, wheat straw) with both rumen and hindgut inocula. Hindgut fermentation revealed lower total gas and methane production and hydrogen recovery. The DM and NDF degradability of treated ®brous materials was signi®cantly higher then of untreated ones for both rumen and hindgut inocula. The DM degradability of CA was higher as compared to CC. However, unlike the other substrates both celluloses showed similar DM degradability values with rumen and hindgut inocula. Total gas and VFA production was in correlation with DM degradability. Characteristic for all ®brous materials was a high mol% of propionate and a low mol% of butyrate with both inocula as compared to meadow hay. The results con®rmed high signi®cance of the treatment of
beech sawdust and wheat straw. The results con®rmed also a higher fermentation activity of the rumen as compared to the hindgut. Reductive acetogenesis in the hindgut content instead of methanogenesis may increase the energetic yield from VFA per fermented substrate and to the same extent also the energetic yield in the host animal.
Acknowledgements
The study was supported by funds from the Grant Agency for Science of the Slovak Academy of Sciences (2/1320).
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