Gas production from straw incubated in vitro with different levels of purified carbohydrates

Animal Feed Science and Technology 101 (2002) 1–15

Gas production from straw incubated in vitro with different levels of purified carbohydrates M. Fondevila∗ , A. BarriosUrdaneta1 , J. Balcells, C. Castrillo a

Departamento de Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain

Received 7 November 2001; received in revised form 30 May 2002; accepted 19 July 2002

Abstract The effect of the type (starch (S); cellulose (C) and pectin (P)) and level (0–4 mg/ml) of added carbohydrate on gas production from ammoniated straw (AS) at a fixed pH (6.7) was studied in vitro in three incubation runs. The S, C and P were also incubated without AS at levels of 1, 2.5 and 4 mg/ml. On a dry weight basis, gas produced from P as the only substrate was higher than S, and from S higher than C up to 12 h of fermentation (P < 0.05). From 24 to 36 h, gas volume from S was higher than P, with no differences after 48 h. Gas produced from AS in mixed incubations was estimated by subtraction of the carbohydrate contribution, calculated by linear regression. Gas volume from AS was higher with P than with S or C at all times (P < 0.05). Proportion of acetate was higher, and propionate lower, in P than in S or C media (P < 0.05). Addition of C or S reduced gas volume from AS compared with level 0 from 24 or 30 h onwards, respectively (P < 0.05). However, inclusion of P increased linearly gas production until 8 h (P < 0.01). In vitro, carbohydrates affected straw fermentation at an optimum pH, being negative with starch and cellulose from 24 h onwards. A positive effect of pectin on straw fermentation was observed in the first 8 h of incubation, and no effect was seen thereafter. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Straw fermentation; Carbohydrate addition; In vitro gas production

1. Introduction The inclusion of readily digestible carbohydrates in forage based diets for ruminants can restrict microbial digestion of structural polysaccharides (Mertens and Loften, 1980; Mould et al., 1983; Fondevila et al., 1994). This effect is caused by a shift in rumen environmental conditions, making them unfavourable for microbial fibrolysis. It has been stated that rumen ∗ Corresponding author. Tel.: +34-976-761660; fax: +34-976-761590. E-mail address: [email protected] (M. Fondevila). 1 Present address: Facultad de Agronom´ıa, Universidad del Zulia, Maracaibo, Venezuela.

0377-8401/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 0 2 ) 0 0 1 8 3 - 9

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acidification of pH to below 6.2–6.0 is the main factor causing this effect (Stewart, 1977; Hiltner and Dehority, 1983). However, some authors (Mould et al., 1983; Piwonka and Firkins, 1993) have speculated that readily digestible carbohydrates can slow, or reduce, cell wall degradation even at optimum rumen pH. Grant and Mertens (1992), incubating cellulosic sources in vitro alone or with 30% corn starch at different pH (5.8, 6.2 and 6.8) concluded that the negative effect of pH on fibre digestion is enhanced by starch inclusion. Both source and level of carbohydrate inclusion may cause this effect and their differences determine the magnitude of the restriction of cell wall fermentation. In vitro, Barrios Urdaneta et al. (2000) showed higher straw cell wall digestion when supplemented with pectin versus soluble sugars or starch at 1:0.55 ratio, even with no differences in medium pH. This effect was mainly attributed to higher bacterial adhesion to cell wall particles at 8 and 12 h of incubation. Fondevila et al. (1994) have also observed higher rumen disappearance of straw when supplemented with sugar beet pulp, a source of highly digestible structural carbohydrates, compared with barley grain as a source of starch. Levels of supplementation of forage diets below 0.15–0.20 have resulted in no effect, or even an increase, in forage fermentation (Silva and Ørskov, 1988), but microbial fibre digestion is generally depressed when carbohydrates are added at over 0.30 of diet (Stewart et al., 1979; Henning et al., 1980). This work compares the effect of three purified carbohydrates with different chemical characteristics included in the medium at different levels on the pattern of in vitro microbial fermentation of straw, at an optimum pH for fibrolytic activity. 2. Materials and methods 2.1. Experimental design The Menke and Steingass (1988) gas production technique was used. Anhydrous ammonia-treated barley straw (AS) was incubated alone or with soluble potato starch (S; Probus), pectin from citrus pulp (P; Sigma) or Sigmacell 20 cellulose (C; Sigma) as supplements. Chemical composition of AS (g/kg dry matter, DM) was: organic matter (OM), 937; neutral detergent fibre (NDF), 782; acid detergent fibre (ADF), 486; acid detergent lignin (ADL), 54; total nitrogen, 13.7. Prior to incubation, AS was ground in a hammer mill to a maximum particle size of 1 mm. The effect of carbohydrates on AS fermentation was studied in three consecutive runs of incubation, comparing carbohydrates in pairs (S versus P; S versus C and P versus C). In addition, another two incubation runs with the three carbohydrates, but without AS, were also completed. Each incubation run included a total of 22 calibrated 100 ml glass syringes, with two blanks of inoculum and two syringes with a standard straw (tested in triplicate in seven consecutive incubation runs) on each run. There were no incubations where differences between the observed mean gas production of the standard with the mean standard value was over 10%. 2.2. Experimental procedures Approximately 200 mg DM of AS were included in each syringe, that were filled with 30 ml of incubation medium, as specified in González Ronquillo et al. (1998). Media

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included 33% rumen fluid from two cannulated sheep given 900 g per day of a 50:50 alfalfa hay:barley straw diet. Two different batches of incubation media were prepared on each run, one for each compared carbohydrate, which was added in increasing amounts to get 1–4 mg/ml medium, or 15, 30, 45 and 60% (w/w) of AS. Another two syringes with only AS were also included (level 0). In the runs without AS (i.e. runs 1 and 5), the three carbohydrates were included at 1, 2.5 and 4 mg/ml media. On each run, all treatments were incubated in duplicated syringes. In all cases, initial pH of media was adjusted to 6.7. Gas production was measured by piston displacement after 2, 4, 6, 8, 12, 24, 30, 36, 48, 60, 72 and 96 h of incubation. Once the incubation was finished, the pH of syringe contents was measured immediately, and then filtered (45 ␮m pore size), recovering both liquid and solid residue. A 4 ml sample of the filtrate was mixed with 1 ml solution of phosphoric acid (2% v/v) and methyl-valeric acid (2 mg/ml), and frozen for volatile fatty acid (VFA) analysis. The pH electrode was rinsed over the solid residue, and this was washed thoroughly with warm distilled water and dried for 48 h at 65 ◦ C to estimate DM disappearance (DMd) and relative gas yield (RGY: millilitres of gas from straw after 96 h/g of DMd), as in González Ronquillo et al. (1998). 2.3. Calculations and chemical analysis Once corrected for the average blank, gas production from straw for each incubation time was estimated by subtracting the carbohydrate contribution (average from runs 1 and 5) from the total gas volume observed in the mixed substrate syringes, and expressed per unit of initial straw DM weight. The volume of gas corresponding to the fermentation of each level of inclusion of S, P and C was estimated from the two runs where only carbohydrates were incubated, by linear regression of the volume produced over their level of inclusion. In all cases, the pattern of gas production for every syringe was adjusted to the model of France et al. (1993): √ √ y = A{1 − exp[−b(t − T ) − c( t − T )]} where y represents the cumulative gas production (ml), t the incubation time (h), A the asymptote (total gas; ml), T the lag time (h), and b and c are the rate constants (h−1 and h−1/2 , respectively). The fractional degradation rate (µ (h−1 )) is considered to vary with time according to µ=

b+c √ , 2 t

t ≥T

Analysis for total N was performed by the Kjeldahl method, and NDF, ADF and ADL content of AS were determined following Van Soest et al. (1991), without using sodium sulphite and without ␣-amylase, and expressed without residual ash. Analysis of VFA was performed by gas–liquid chromatography (Jouany, 1982). 2.4. Statistical analysis Gas production from straw was statistically analysed by ANOVA for each incubation time. Carbohydrates were contrasted in pairs (S versus P; S versus C and P versus C) considering

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each syringe as the experimental unit and without including level 0 in the comparison. The effects of type and level (15, 30, 45 and 60%) of carbohydrate inclusion, and their interaction, were contrasted against the residual (8 degrees of freedom (d.f.)). For a further study of the level of inclusion, data for the two runs in which each carbohydrate was incubated were analysed together. The level of inclusion (0, 15, 30, 45 and 60%) was contrasted against the residual (14 d.f.), with the incubation run as a block. Three orthogonal contrasts were also planned in this case (i.e. no versus addition; linear effect; quadratic effect). Gas production from the carbohydrates alone were also studied, contrasting type, level of inclusion and their interaction against the residual (26 d.f.), with the incubation run as a block. In all cases, differences among experimental treatments were contrasted by Tukey’s t-test.

3. Results There were no differences (P > 0.10) in final pH (between 6.54 and 6.65 after 96 h of fermentation) among carbohydrates or their inclusion levels. 3.1. Gas production from the added carbohydrates When the carbohydrates were incubated as the only substrate, the volume of gas produced increased linearly (P < 0.001) with the level of inclusion (data not presented). Neither the level of inclusion nor the interaction type x level reached significant differences at any incubation time (P > 0.05). Therefore, gas production from S, P and C as the only substrate is expressed on initial weight basis in Fig. 1. Up to 12 h of incubation, the volume of gas

Fig. 1. Average gas production pattern from the supplements (starch (䊏); cellulose (䊉); pectin (䉱)) when incubated as the only substrate, expressed per unit of incubated dry matter (n = 12). Upper bars show S.E.M.

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Fig. 2. Average gas production pattern from ammonia-treated straw when supplemented with starch (䊏) or pectin (䉱), once subtracted their contribution to the total gas produced (n = 8). Upper bars show S.E.M.

produced was lower from C than S and P (P < 0.05). The gas production from P was also higher (P < 0.05) than S from 4 to 12 h of incubation. At 24 h of incubation, the volume of gas produced from S was higher than C and P (P < 0.05), and at 30 and 36 h it was also higher than with P (P < 0.05). From Fig. 1, it is evident that S and C plateaued about 36 h of incubation, whereas with P it occurred at 48 h. 3.2. Effect of the type of carbohydrate on straw fermentation Figs. 2–4 compare the effect of the carbohydrates studied in pairs (S versus P; S versus C and P versus C, respectively) on the pattern of gas produced in vitro by microbial fermentation of straw. Values presented were obtained by subtraction of the carbohydrate contribution to the gas volume, estimated by linear regression from the incubation runs with the carbohydrates as the only substrate, from the total volume produced in the syringes with mixed substrate. Each point shows the average gas volume for the four levels of inclusion of each carbohydrate in the incubated syringes (n = 8). When the effects of S and P were compared (Fig. 2), gas production from AS was higher with P than with S (P < 0.001) at any incubation time, except for 8 (P < 0.05) and 12 h, when with S was higher than P (P < 0.05). The interaction type of carbohydrate x level up to 6 h of incubation (P < 0.05) indicates that these differences were not observed at 15% inclusion. The DMd after 96 h of incubation tended to be higher with S than P (689 mg/g versus 671 mg/g; P < 0.10), whereas the RGY was higher with P (321 ml/g versus 358 ml/g DM disappeared for S and P; P < 0.01). The curve parameters obtained when gas volumes for carbohydrates comparison from all incubations, adjusted to the equation proposed by

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Fig. 3. Average gas production pattern from ammonia-treated straw when supplemented with starch (䊏) or cellulose (䊉), once subtracted their contribution to the total gas produced (n = 8). Upper bars show S.E.M.

France et al. (1993), are in Table 1 with the fractional rates of gas production estimated at 12, 24 and 48 h of incubation. Total volume of gas produced (A) was higher and lag time (T) lower with P than S (P < 0.05). Fractional fermentation rates were also higher for P than S at 12 h (P < 0.05) and, not significantly, at 24 and 48 h (P < 0.10).

Fig. 4. Average gas production pattern from ammonia-treated straw when supplemented with cellulose (䊉) or pectin (䉱), once subtracted their contribution to the total gas produced (n = 8). Upper bars show S.E.M.

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Table 1 Average coefficients of the curves adjusted from the gas production pattern of straw depending on the type of supplement, once subtracted the supplement contribution T

µ12

µ24

µ48

0.013 0.013 0.0217

2.16 1.24 0.242∗

0.047 0.056 0.0024∗

0.046 0.055 0.0030

0.046 0.055 0.0035

0.034 0.075 0.0064∗∗

0.025 −0.176 0.0333∗∗

3.03 3.00 0.477

0.037 0.049 0.0030∗

0.036 0.057 0.0037∗∗

0.035 0.062 0.0044∗∗

0.084 0.062 0.0043∗∗

−0.273 −0.087 0.0196∗∗∗

3.23 1.82 0.279∗∗∗

0.044 0.049 0.0026

0.056 0.053 0.0029

0.064 0.055 0.0033

Supplement

A

b

Starch Pectin S.E.M.

44.2 48.2 1.02∗

0.045 0.054 0.0049

Starch Cellulose S.E.M.

44.9 41.4 1.92

Cellulose Pectin S.E.M.

41.6 47.9 1.42∗∗

c

A: total gas (ml/200 mg DM); b, c: rate constants (h−1 and h−1/2 , respectively); µ: fractional degradation rate (h−1 ); S.E.M.: standard error of means. ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001.

Straw contribution to the gas volume when S or C were added (Fig. 3) did not differ with the type of carbohydrate, except at 24 h of incubation, when C induced a higher gas production from straw than S (P < 0.05). There were no differences between carbohydrates in DMd and RGY (599 mg/g versus 603 mg/g and 358 ml/g versus 344 ml/g DM for S and C, respectively; P > 0.05). When S and C were compared (Table 1), there were differences between them in the rate constants b and c, that were manifested in higher fractional rates for C (P < 0.05). The pattern of gas produced from AS with P or C is in Fig. 4. Higher gas volumes were recorded with P until 72 h incubation (P < 0.05). These differences between P and C were not the same for all levels of inclusion, since at 15% inclusion there were differences only at 12 and 24 h (interaction type of carbohydrate x level; P < 0.05). Straw DMd was lower with C than P (579 mg/g versus 619 mg/g; P < 0.01), and there were no differences in RGY (359 ml/g versus 372 ml/g DM; P > 0.05). As shown in Table 1, treatment P also promoted a higher A (P < 0.01) and a lower T (P < 0.001) than C, and rate constants b and c were higher and lower, respectively, with P than C (P < 0.01). However, differences in the fractional rate were not significant (P > 0.05). It is noticeable that in all incubation runs, straw fermentation rate with C increased with time, whereas no changes were observed with both S and P. Table 2 shows average total VFA values after 96 h of incubation and molar acetate, propionate and butyrate proportions for each treatment on each incubation run. There were no differences in total VFA concentration between S and P addition, but acetate proportion was lower and that for propionate and butyrate higher in the former (P < 0.01). In contrast, total VFA concentration and propionate proportion were higher in C than in S (P < 0.01), whereas butyrate proportion was higher with S. When comparing P and C, acetate proportion was higher in the former, whereas that of propionate was higher with C (P < 0.01).

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Table 2 Average total volatile fatty acid (VFA) concentration (mmol/l) and molar acetate, propionate and butyrate proportions (%) after 96 h of in vitro incubation of ammonia-treated straw supplemented with starch, cellulose or pectin Total VFA

Acetate

Propionate

Butyrate

Starch Pectin S.E.M.

69.5 66.2 1.74

66.9 71.2 0.18∗∗

20.9 17.7 0.17∗∗

7.2 6.6 0.06∗∗

Starch Cellulose S.E.M.

68.3 82.9 2.32∗∗

66.9 66.9 0.39

20.1 22.4 0.19∗∗

8.4 6.5 0.14∗∗

Cellulose Pectin S.E.M.

70.6 66.0 3.20

68.2 72.4 0.57∗∗

23.2 17.8 0.21∗∗

5.1 6.2 0.43

S.E.M.: standard error of means. ∗∗ P < 0.01.

3.3. Effect of the level of inclusion on straw fermentation Figs. 5–7 show patterns of gas production from AS with 0, 15, 30, 45 and 60% S, P or C, respectively, in two runs of incubation for each carbohydrate. There were no differences among the different levels of S (Fig. 5) up to 30 h of fermentation. From 30 h onwards, gas production for level 0 was higher than the carbohydrate-including treatments, and differences between levels 15 and 60 were also apparent (P < 0.05). No linear or quadratic

Fig. 5. Average gas production pattern from ammonia-treated straw, alone (䊊) or supplemented with 15 ( ), 30 (䊉), 45 (䊏) or 60 (䉱)% starch in two runs of incubation, once subtracted the contribution of the supplement to the total gas produced (n = 4).

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Fig. 6. Average gas production pattern from ammonia-treated straw, alone (䊊) or supplemented with 15 ( ), 30 (䊉), 45 (䊏) or 60 (䉱)% cellulose in two runs of incubation, once subtracted the contribution of the supplement to the total gas produced (n = 4). Upper bars show S.E.M.

Fig. 7. Average gas production pattern from ammonia-treated straw, alone (䊊) or supplemented with 15 ( ), 30 (䊉), 45 (䊏) or 60 (䉱)% pectin in two runs of incubation, once subtracted the contribution of the supplement to the total gas produced (n = 4). Upper bars show S.E.M.

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Table 3 Coefficients of the curves adjusted from the gas production pattern of straw depending on the level of supplementation, once subtracted the supplement contribution A

b

c

T

µ12

µ24

µ48

Starch 0%a 15% 30% 45% 60% S.E.M.

43.2 46.0 43.7 45.0 43.6 1.81

0.054 0.041 0.049 0.044 0.024 0.0121

−0.126 0.002 −0.025 0.000 0.099 0.0378

2.44 2.58 2.04 2.76 3.01 0.448

0.035 0.041 0.045 0.044 0.038 0.0067

0.041 0.041 0.046 0.044 0.034 0.0083

0.045 0.041 0.047 0.044 0.031 0.0094

Cellulose 0% 15% 30% 45% 60% S.E.M.

48.31 43.98 39.55 43.27 39.24 1.742

0.081 0.076 0.083 0.068 0.089 0.0058

−0.250 −0.174 −0.252 −0.174 −0.299 0.0472

3.32 3.34 4.01 1.94 3.19 0.630

0.045 0.051 0.047 0.043 0.046 0.0018

0.056 0.058 0.058 0.050 0.059 0.0017

0.063 0.064 0.065 0.055 0.068 0.0026

Pectin 0% 15% 30% 45% 60% S.E.M.

44.86 44.82 46.24 50.00 51.10 3.372

0.060 0.087 0.073 0.048 0.024 0.0114

−0.175 −0.198 −0.125 0.050 0.126 0.0455

2.12 2.90 1.24 1.00 0.99 0.471

0.035 0.059 0.054 0.055 0.042 0.0062

0.042 0.067 0.060 0.053 0.037 0.0075

0.047 0.073 0.064 0.051 0.033 0.0086

A: total gas (ml/200 mg DM); b, c: rate constants (h−1 and h−1/2 , respectively); µ: fractional degradation rate (h−1 ); S.E.M.: standard error of means. a Percentage of ammonia-treated straw (w/w).

trends were observed at any incubation time (P > 0.05). Adjusted curve parameters for the increasing levels of carbohydrates are in Table 3. Differences between level 0 and the levels including S were only detected for rate constant c (P < 0.01). When AS was supplemented with C (Fig. 6), there were differences between level 0 and treatments including cellulose at 6 and 8 h of incubation and from 24 h onwards (P < 0.05). No differences were detected among the levels of inclusion of cellulose at any control time. For C, coefficient A was higher (P < 0.05) in level 0 than in the other treatments (Table 3), and linear and quadratic trends were observed for the fractional fermentation rate at 24 h of incubation (P < 0.05). In contrast to S and C inclusion, P increased the proportion of gas volume produced from straw compared with AS alone (Fig. 7) up to 8 h of fermentation (P < 0.01). This effect was linear as the proportion of pectin increased from 0 to 8 h (P < 0.01), and also appeared to be at 30 and 36 h (P < 0.05), but in this case the effect of the level of inclusion was not significant. There were no differences among the examined levels of P from 48 h onwards (P < 0.05). There was a linear trend towards a decrease in lag time T (Table 3) when increasing levels of P were added to AS (P < 0.05). There was also a decrease in the rate constant b and an increase in the rate constant c (P < 0.05) that were reflected by a trend (P < 0.10) to a decrease in fractional degradation rate at 36 and 48 h as the level of pectin increased.

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Table 4 Average total volatile fatty acid (VFA) concentration (mmol/l) and molar acetate, propionate and butyrate proportions (%) after 96 h of in vitro incubation of ammonia-treated straw supplemented with different levels of starch, cellulose or pectin Total VFA

Acetate

Propionate

Butyrate

Starch 0%a 15% 30% 45% 60% S.E.M.

51.9 c 55.2 bc 68.8 ab 74.8 a 76.9 a 5.13∗∗∗

68.7 a 67.1 ab 67.6 ab 67.0 ab 66.0 a 0.72∗

18.5 c 19.8 bc 19.8 bc 20.7 ab 21.7 a 0.47∗∗

7.5 7.9 7.8 7.8 7.8 0.26

Cellulose 0% 15% 30% 45% 60% S.E.M.

51.4 b 74.2 a 71.6 a 75.4 a 84.9 a 6.05∗∗∗

69.0 ab 71.8 a 67.4 b 66.5 b 65.8 b 1.11∗∗∗

18.7 a 19.4 a 22.2 b 23.7 c 24.9c 0.42∗∗∗

7.3 4.7 6.3 6.1 5.8 0.78

Pectin 0% 15% 30% 45% 60% S.E.M.

50.3 a 58.1 bc 60.8 bc 69.0 ab 80.9 a 4.57∗∗∗

68.2 c 70.8 b 71.1 b 72.1 ab 73.9 a 0.66∗∗∗

19.4 a 18.3 b 18.0 b 17.6 bc 16.7 c 0.30∗∗∗

7.1 a 6.5 ab 6.6 ab 6.4 ab 6.0 b 0.23∗∗

For each comparison, different letters indicate significant differences (P < 0.05). S.E.M.: standard error of means. a Percentage of ammonia-treated straw (w/w). ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001.

No differences were observed for RGY in response to increasing levels of S, P or C, and differences between level 0 and the further levels of inclusion in DMd were only detected for C (P < 0.05), showing in this reduction a trend to linearity (P < 0.10). Carbohydrate addition increased VFA production compared with level 0 (P < 0.01; Table 4). For all carbohydrates, total VFA increased linearly with their level of inclusion (P < 0.001). Acetate proportion decreased linearly with the level of S and C, at the time propionate increased (P < 0.001). In contrast, with P acetate increased as both propionate and butyrate decreased (P < 0.001). There was no quadratic trend at any case.

4. Discussion Incubation of starch, cellulose and pectin as the only substrates indicates the higher potential for microbial fermentation of pectin in the first 12 h of incubation. Chesson and Monro (1982) have already pointed out the rapid fermentation of pectins. However, its

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rate of gas production decreased afterwards, and reached its plateau later than both starch and cellulose. Starch and cellulose evolved similarly, but the lag time for the latter seemed longer. Beuvink and Spoelstra (1992) observed a higher volume and rate of gas production with rice starch than with crystalline cellulose, but in our case the amorphous structure of Sigmacell cellulose might be expected to result in smaller differences. It is noteworthy that these authors also observed higher total VFA production and propionate proportion with cellulose as substrate than with starch. The straw contribution to the total volume of gas produced in mixed substrate treatments (incubation runs 2–4) was calculated by subtracting from the total gas the amount that was expected from the fermentation of the carbohydrate added in the incubation. This calculation is based on the assumption that straw does not affect supplement fermentation characteristics, whereas an effect of the supplement can be expected. Other authors have applied similar subtraction calculations in vivo (Chenost, 1987) and also to in vitro gas production studies, for example to estimate NDF contribution to total plant gas production (Stefanon et al., 1996) or the contribution of added organic acids in silage fermentation (Deaville and Givens, 1998). In order to minimise the error of correction, the volume of gas produced by any specific amount of S, C or P, fermentation was estimated by linear regression of the gas produced on the levels of inclusion of each carbohydrate at a given incubation time, since gas production depends linearly on the amount of substrate (Menke and Steingass, 1988). For our experiment, carbohydrates were chosen according to expected different effects on microbial fermentation: starch because of its negative effect on microbial fibre digestion; cellulose because in some situations it may be preferred by cellulolytic bacteria thus reducing straw digestion; and pectin because, although being an easily fermentable carbohydrate, it negatively affects the rumen environment in a lesser extent. Straw fermentation was depressed by inclusion in the medium of starch and cellulose, as shown when comparing AS as the only substrate with media including these carbohydrates. However, this was not the case for pectin, as it did not reduce straw fermentation from 24 h onwards, but even increased gas production from straw through 24 h. This was also evident in a lower magnitude of the T coefficient and the fractional degradation rate at 12 h compared with AS alone (Table 3). Furthermore, the volume of gas produced from straw per unit of disappeared DM was also higher with P than S, suggesting a more efficient utilisation of the energy from straw with the former. Obviously, the magnitude of these effects is to a large extent linked to the amount of added carbohydrate, especially when it is over 25–30% inclusion. The increase in gas production from straw responded linearly to the level of pectin up to 8 h, whereas the lack of a significant linear response with starch and cellulose was caused by the absence of differences among levels 15, 30 and 45% of starch inclusion, and among all levels of cellulose. From the comparison between starch and cellulose (Fig. 3), it can be seen that there were no differences in the magnitude of their effect on straw fermentation. The negative effect of starch on forage fermentation has been widely documented (Silva and Ørskov, 1988; Fondevila et al., 1994), and mainly attributed to a drop in pH (Hiltner and Dehority, 1983; Stritzler et al., 1998), but in the current experiment pH was constant. Firkins et al. (1991) also observed a reduction in digestion of crystalline cellulose when 30% starch was included in the medium. These authors observed higher microbial growth, and also higher adhesion to particles in the supplemented treatments, that was not necessar-

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ily associated with a higher enzymatic activity against structural polysaccharides (Barrios Urdaneta et al., 2000). Other factors may also be involved in depression of straw digestion, such as competition for growth factors favourable to the more metabolically active amylolytic bacteria (Russell, 1984) or a depression in Fibrobacter succinogenes cellulase activity in presence of high glucose concentrations (Huang and Forsberg, 1990). A shift of F. succinogenes to a preferential utilization of starch or soluble sugars instead of cellulose have also be suggested (Huang and Forsberg, 1990; Khalili and Huhtanen, 1991), although no starch digestion with this bacteria was observed by Bryant and Doetsch (1954). Implicated mechanisms in the negative effect of cellulose may be related to a feed-back effect of glucose and cellobiose on cellulolytic enzymes (Huang and Forsberg, 1990) and to a preferred utilisation of purified cellulose instead of the less accessible chemical structure of straw fibre. In contrast, Silva and Ørskov (1988) suggested that the inclusion of a good quality forage in the diet increases rumen degradation of straw. There is not much information regarding the effect of pectin inclusion in diet on fibre digestion in the rumen, but both a higher bacterial adhesion to straw particles and higher cellulase activity have been observed in our laboratory (Barrios Urdaneta et al., 2000). Either a physical effect of pectin, providing a protective environment for microorganisms adhered to forage particles, or a chemical action by fixing cations, especially Ca++ , thus neutralising repellent ionic forces and favouring adhesion (Roger et al., 1990) may be involved. Moss (1994) suggested that sugar beet pulp, as a source of pectins, might increase microbial colonisation of fibrous particles when supplementing straw diets. 5. Conclusions Results suggest that addition of carbohydrates such as starch and cellulose negatively affects in vitro microbial fermentation of straw, even at an optimum pH. This effect was evident after 24 h of in vitro incubation, reducing the proportional contribution of straw to overall gas production to a similar extent for both carbohydrates. Although this depression was not linear, maximum differences were observed in both cases between the straw alone and the maximum level of inclusion. Gas production from straw linearly increased with the level of pectin in the first 24 h of fermentation. This suggests that, in practical conditions, addition of pectin rich feeds to straw based diets may have a positive associative effect on rumen utilisation of straw. Acknowledgements This work was financed by the Project PCA 1694 (Diputación General de Aragón, Spain). The doctoral stage of Dr. Barrios Urdaneta was supported by CONICIT (Venezuela). References Barrios Urdaneta, A., Fondevila, M., Balcells, J., Dapoza, C., Castrillo, C., 2000. In vitro microbial digestion of straw cell wall polysaccharides in response to supplementation with different sources of carbohydrates. Aust. J. Agric. Res. 51, 393–399.

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Beuvink, J.M.W., Spoelstra, S.F., 1992. Interactions between substrate, fermentation end-products buffering system and gas production upon fermentation of different carbohydrates by mixed rumen microorganisms in vitro. Appl. Microbiol. Biotechnol. 37, 505–509. Bryant, M.P., Doetsch, R.N., 1954. A study of actively cellulolytic rod-shaped bacteria of the bovine rumen. J. Dairy Sci. 37, 1176–1183. Chenost, M., 1987. Influence de la complémentation sur la valeur alimentaire et l’utilisation des mauvais foins et des pailles par les ruminants. In: Demarquilly, C. (Ed.), Les fourrages secs: récolte, traitement, utilisation. INRA, Paris, pp. 183–198. Chesson, A., Monro, J.A., 1982. Legume pectin substances and their degradation in the ovine rumen. J. Sci. Food Agric. 33, 852–859. Deaville, ER, Givens, DI, 1998. Investigation on direct gas production from silage fermentation acids. In: Proceedings of the British Society of Animal Science, Scarborough, March 1998, p. 64. Firkins, J.L., Bowman, J.G.P., Weiss, W.P., Naderer, J., 1991. Effects of protein, carbohydrate and fat sources on bacterial colonization and degradation of fiber in vitro. J. Dairy Sci. 74, 4273–4283. Fondevila, M., Castrillo, C., Guada, J.A., Balcells, J., 1994. Effect of ammonia treatment and carbohydrate supplementation of barley straw on rumen liquid characteristics and substrate degradation by sheep. Anim. Feed Sci. Technol. 50, 137–155. France, J., Dhanoa, M.S., Theodorou, M.K., Lister, S.J., Davies, D.R., Isac, D., 1993. A model to interpret gas accumulation profiles associated with in vitro degradation of ruminant feeds. J. Theor. Biol. 163, 99–111. González Ronquillo, M., Fondevila, M., Barrios Urdaneta, A., Newman, Y., 1998. In vitro gas production from buffel grass (Cenchrus ciliaris L.) fermentation in relation to the cutting interval, the level of nitrogen fertilisation and the season of growth. Anim. Feed Sci. Technol. 72, 19–32. Grant, R.J., Mertens, D.R., 1992. Influence of buffer pH and raw corn starch addition on in vitro fiber digestion kinetics. J. Dairy Sci. 75, 2762–2768. Henning, P.A., Linden, Y., Mattheyse, M.E., Nauhaus, W.K., Schwartz, H.M., 1980. Factors afffecting the intake and digestion of roughage by sheep fed maize straw supplemented with maize grain. J. Agric. Sci. 94, 565–573. Hiltner, P., Dehority, B.A., 1983. Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria. Appl. Environ. Microbiol. 46, 642–648. Huang, L., Forsberg, C.W., 1990. Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85. Appl. Environ. Microbiol. 56, 1221–1228. Jouany, J.P., 1982. Volatile fatty acid and alcohol determination in digestive contents, silage juices, bacterial cultures and anaerobic fermentor contents. Sci. des Aliments 2, 131–144. Khalili, H., Huhtanen, P., 1991. Sucrose supplements in cattle given grass silage-based diet. Part 2. Digestion of cell wall carbohydrates. Anim. Feed Sci. Technol. 33, 263–273. Menke, K.H., Steingass, H., 1988. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Dev. 28, 7–55. Mertens, D.R., Loften, J.R., 1980. The effect of starch on forage fiber digestion in vitro. J. Dairy Sci. 63, 1437–1446. Moss, A.R., 1994. Methane production by ruminants. Literature review of: I. Dietary manipulation to reduce methane production and II. Laboratory procedures for estimating methane potential of diets. Nutr. Abstr. Rev., Series B 64 (12), 785–806. Mould, F.L., Ørskov, E.R., Mann, S.O., 1983. Associative effects of mixed feeds. Part 1. Effect of type and level of supplementation and the influence of the rumen fluid pH on cellulolysis in vivo and dry matter digestion of various roughages. Anim. Feed Sci. Technol. 10, 15–30. Piwonka, E.J., Firkins, J.L., 1993. Effect of glucose on fiber digestion and particle-associated carboxymethylcellulase activity in vitro. J. Dairy Sci. 76, 129–139. Roger, V., Fonty, G., Komisarczuk-Bony, S., Gouet, P., 1990. Effects of physicochemical factors on the adhesion to cellulose avicel of the ruminant bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes subsp. Succinogenes. Appl. Environ. Microbiol. 56, 3081–3087. Russell, J.B., 1984. Factors influencing competition and composition of the rumen bacterial flora. In: Gilchrist, F.M.C., Mackie, R.I. (Eds.), Herbivore Nutrition in the Subtropics and Tropics. The Science Press, Pretoria, pp. 313–345. Silva, A.T., Orskov, E.R., 1988. Fiber degradation in the rumen of animals receiving hay, untreated or ammonia-treated straw. Anim. Feed Sci. Technol. 29, 251–264.

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