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Use of the in vitro cumulative gas production technique for pigs: An examination of alterations in fermentation products and substrate losses at various time points¹

Animal Nutrition Group, Wageningen University and Research Centre, Wageningen, The Netherlands

	Abstract

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An experiment was conducted to examine changes in VFA and ammonia concentrations at different time points using 4 fermentable carbohydrate-rich feed ingredients as substrates and feces of unweaned piglets as inoculum. Fecal inoculum was collected, pooled, and mixed from 9 specially raised (no creep feed or antibiotics) crossbred piglets at 3 wk of age. Inulin, lactulose, molasses-free sugar beet pulp, and wheat starch were used as substrates and were fermented in vitro for 72 h (3 replicates per substrate). Cumulative gas production was measured as an indicator of the kinetics of fermentation. In addition, 3 bottles of substrate per time point with similar contents (amounts of substrate, inoculum, and media) were incubated but were allowed to release their gas throughout incubation. For these latter bottles, fermentation fluid was sampled at incubation time points including every hour between 1 and 24 h and at 48 h, and fermentation end products (VFA, lactate, and ammonia) and OM disappearance were measured. Dry matter and ash were analyzed from the postfermentative samples. The pH of the contents from these bottles was also recorded. The correlation in time between fermentation end products and cumulative gas produced was determined. The results showed that the prolongation of fermentation to 72 h, especially in the case of fast-fermenting inulin and lactulose, may lead to a different end product profile (P < 0.001) compared with the profile observed at the time at which most of the substrate has disappeared. Therefore, we concluded that the fermentation product profile at the end of in vitro fermentation at a specific time point cannot be used to compare fermentability of carbohydrate sources with different fermentation kinetics in terms of gas production.

Key Words: fermentable carbohydrate • fermentation • in vitro gas production

	INTRODUCTION

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The basic principle of all gas production techniques is that the in vitro fermentation of feeds by microorganisms is accompanied by the production of gas. The gas is formed directly by microbial fermentation of the substrate as well as indirectly by release of carbon dioxide caused by production of VFA from the bicarbonate buffer, which is often used in these techniques (Van Soest, 1994). Therefore, the amount of gas produced depends on both the amount of substrate fermented and the amount and molar proportions of the VFA produced (Davies et al., 2000). However, it is very difficult to separate direct vs. indirect gas production. Based on the fact that both are directly related to the fermentation of a substrate, the gas production measured at each time point can be considered as an index for fermentation activity (Groot et al., 1996).

In ruminants, the gas production technique is mainly used for forage evaluation (Williams, 2000). The cumulative gas production technique of Theodorou et al. (1994), which was further automated by Davies et al. (1995), has been proposed for use in studies concerning nonruminant fermentation by Williams et al. (2001) and by Bauer (2002). In the standard technique used mainly for feed evaluation, the fermentation product profile (usually VFA and ammonia) is analyzed by sampling at the end of a standard fermentation time.

The current study aimed to evaluate 1) whether the fermentation product profile at the end of the specific fermentation time is representative of the product profile present at the time when the substrate has been almost fully fermented and 2) whether the in vitro fermentation product pattern at different time points correlates with cumulative gas produced at each time point.

	MATERIALS AND METHODS

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Experimental Design

The study was conducted using the Automated Pressure Evaluation System (APES; Davies et al., 2000). Nine 3-wk-old unweaned piglets from 3 litters were used as fecal donors, and the feces were collected, pooled, and mixed to have both a representative microbial population and sufficient material as inoculum. All of the procedures involving animals were conducted in accordance with the Dutch law on experimental animals and were approved by the Wageningen University Animal Experimental Committee.

Animals

The 9 crossbred piglets from 3 different litters (3 from each litter) had free access to sow’s milk and did not receive any creep feed or antibiotics before the experiment. During the complete experimental period, piglets remained with their sow and littermates on the farm.

Substrates

Four carbohydrate-rich ingredients (inulin, lactulose, molasses-free sugar beet pulp, and wheat starch) were used as substrates. All substrates were obtained from commercial suppliers. The air-dried substrates were ground to pass a 1-mm sieve before being used for the in vitro experiment. The substrates were known to have different fermentation kinetics and were chosen based on previous studies in our laboratory (Awati, 2005).

Inoculum

Feces were collected from the rectum with a gloved finger at the farm. Feces were immediately placed in small containers filled with CO₂. In the laboratory, sterile anaerobic saline (0.9% NaCl) was prepared. The water was boiled and bubbled with flow of CO₂ prior to making the solution; the solution was then autoclaved to ensure sterility. The equal amounts of feces from each animal were combined (by wet weight) and diluted 1:15 with prewarmed (to 39°C) sterile anaerobic saline. The large dilution was necessary because of the very small quantity of material obtained. This diluted mixture was homogenized for 60 s to ensure proper mixing and was then filtered through a double piece of clean cheesecloth (16 threads/cm in each direction). The resultant filtrate was used as the inoculum. To maintain anaerobic conditions, all procedures were carried out under a constant stream of carbon dioxide. The fecal matter used was assumed to have a microbial population representative of the large intestinal microflora (Coates et al., 1988; Williams et al., 1998; Bauer et al., 2004).

Four milliliters of inoculum was injected into each fermentation bottle, and then 2 different procedures were followed. For procedure 1, the bottles were immediately attached to the APES (3 replicates per substrate). Each bottle contained ~0.5 g of substrate (as-is basis) and 78 mL of a modified semidefined medium (Lowe et al., 1985). Blanks contained only medium and inoculum. Bottles were incubated at 39°C for 72 h. For procedure 2, in addition to the bottles attached to the APES, 3 bottles per substrate per time point with similar contents (amounts of substrates, inoculum, and medium) were incubated at 39°C. The fermentation was stopped at different time points at every hour between 1 and 24 h and at 48 h by immediately autoclaving the bottles. The caps of these bottles were pierced with a needle to allow gas to escape during the entire incubation. These bottles were sampled for VFA and ammonia analyses. In addition, pH of contents from these bottles was recorded. Organic matter disappearance was determined at each time point.

Analyses

All substrates were analyzed for their DM (ISO 6496:1999; ISO, 1999) and ash (ISO 5984:1978; ISO, 1978) contents. The pH (pH meter, Hanna Instruments, Ijsselstein, The Netherlands) and DM content of the postfermentative samples were determined.

Volatile fatty acid concentrations in the fermentation liquids were analyzed by gas chromatography (Fisons HRGC Mega 2, CE Instruments, Milan, Italy) at 190°C using a glass column fitted with Chromosorb 101. For VFA analysis, the carrier gas was N₂ saturated with methanoic acid, and isocaproic acid was used as an internal standard. Lactic acid concentration of the digesta was analyzed according to the method described by Voragen et al. (1986) using a Jasco HPLC unit (Jasco Benelux BV, Maarssen, The Netherlands) fitted with a Supelcogel HPLC column (30 cm x 7.8 mm i.d.; C-610 H, Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands). Ammonia was determined according to the method described by Searle (1984).

Gas Production Kinetics

The bottles attached to the APES were incubated for 72 h, and gas production kinetics was studied. Data for OM cumulative gas production (mL of gas produced/g of OM weighed into the bottle) were fitted to the monophasic model described by Groot et al. (1996) as follows: G = A/[1 + (C/t)^B], where G = total gas produced (mL), A = asymptotic gas production, B = switching characteristic of the curve, C = time at which one-half of the asymptote had been reached (T_1/2), and t = time (h). The maximum rate of gas production (R_max) and the time at which it occurred (T_max) were calculated according to the following equations (Bauer et al., 2001): R_max = {A x (C^B) x B x [T_max ^(–B–1)]}/{1 + (C^B) x [T_max^(–B)]}² and T_max = C x {[(B – 1)/(B + 1)]^(1/B)}, respectively.

Statistics

Effects of substrate and incubation time (specifically 24, 48, and 72 h) on the fermentation kinetics were tested for significance using a 2-way ANOVA by Proc GLM of SAS (SAS Inst. Inc., Cary, NC). The statistical model was Y = µ + S_i + T_j + (S x T)_ij + _ijk, where Y = parameter to be tested, µ = mean, S_i = effect of substrate i, T_j = effect of incubation time j, (S x T)_ij = interaction, and _ijk = error term. The effect of replicate bottles was tested separately and was not significant (P 0.10) for any of the variables. Therefore, replicate was excluded from the statistical model.

The correlation among time, cumulative gas produced, pH, and production of different fermentation end products was analyzed by Proc Corr of SAS. The existence of a quadratic component in this relationship was tested; however, because it failed to improve the statistical fit over the linear correlation, the linear correlation was selected.

	RESULTS

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DM and Ash

The DM and ash contents, respectively, of the different substrates were inulin (946.1 and 1.9 g/kg), lactulose (703.5 and 0.3 g/kg), sugar beet pulp (902.4 and 67.9 g/kg), and wheat starch (849.8 and 2.4 g/kg).

Cumulative Gas Production Kinetics

Gas production kinetics during 72 h of incubation is shown in Figure 1. The mean values for fitted gas production variables are shown in Table 1. There was no difference (P = 0.21) observed for OM cumulative volume between substrates. However, T_1/2, T_max, and R_max were different (P < 0.001). Inulin had the fastest fermentation among the 4 substrates with the greatest R_max and earliest T_1/2 and T_max, whereas sugar beet pulp had the slowest fermentation with the lowest R_max and the slowest T_1/2 and T_max (Table 1).

View larger version (15K): [in this window] [in a new window]   Figure 1. Gas production kinetics during the 72-h fermentation of 4 carbohydrates [inulin (- - -), lactulose (——), sugar beet pulp (— - —), and wheat starch (— - - —)] by fecal inoculum of suckling piglets.

View larger version (13K): [in this window] [in a new window]   Figure 2. Rate of gas production (mL/h) during the 72-h fermentation of 4 carbohydrates [inulin (), lactulose (), sugar beet pulp (•), and wheat starch (x)] by fecal inoculum of suckling piglets.

Correlation coefficients of fermentation variables and end products with time (for 72 h) and among each other are shown in Table 2. Correlations among the end products and cumulative gas produced with incubation time were different for the different substrates. The cumulative gas produced at different time points showed a positive relationship (P < 0.001) with incubation time. The coefficient of correlation was much lower for the lactulose and inulin compared with wheat starch and sugar beet pulp. Organic matter disappearance during fermentation influenced (P < 0.05) most of the variables, except for lactulose. Total VFA concentration was positively related to cumulative gas produced. The correlation coefficient was increased from inulin (0.77) and lactulose (0.78) to wheat starch (0.86) and sugar beet pulp (0.95). Incubation fluid pH also decreased with incubation time for sugar beet pulp (P < 0.05) and wheat starch (P < 0.001); however, the decrease was not significant for inulin and lactulose.

Table 3 shows fermentation end product profiles at 24, 48, and 72 h. Mean values of fermentation end product concentrations, OM disappearance, and pH showed effects of incubation time, substrates, and their interaction. Substrate (except for lactic acid; P < 0.001) and incubation time (except for lactic and propionic acid; P < 0.01) influenced fermentation end products. However, in the case of lactic acid, pH, and OM disappearance, the substrate x incubation time interaction was significant (P < 0.05). For all substrates incubated, lactic acid had disappeared by 72 h.

	DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The 4 carbohydrate substrates used for the fermentation were known to have different fermentation kinetics (Awati, 2005), which was confirmed in the present experiment (Figure 1; Table 1). Inulin had the fastest fermentation kinetics, and sugar beet pulp had the slowest fermentation kinetics. These differences were essential for an examination of end product kinetics at different time points in relation to maximum substrate disappearance.

According to standard procedures, the samples were collected for the fermentation end products at the end of the specific time interval to compare in vitro fermentability of the different substrates (Deaville and Givens, 2001; DePeters et al., 2003; Guo et al., 2003) or different sources of inoculum (Bauer et al., 2001, 2004). Time intervals vary considerably, although most in vitro incubations have been conducted between 24 and 72 h. The current study evaluated the hypothesis that the fermentation end product profile would change once maximum substrate loss had occurred. Depending on when that loss occurs, it may mean that comparing substrates with very different rates of fermentation at one fixed point is not valid.

Table 2 shows the correlation matrices for the 4 different carbohydrate sources. For the same inoculum, it was observed that the correlation coefficients between the variables were different for different substrates. Specifically, the correlation between the incubation time and cumulative gas produced was significant for all of the substrates, but correlation coefficients were much lower for lactulose (0.60) and inulin (0.52) than for sugar beet pulp (0.94) and wheat starch (0.87; Table 2). Figure 2 shows the rate of gas production at different time points. In the case of lactulose and inulin, the rate of gas production decreased to near zero by 26 and 24 h, respectively. Therefore, it seems that there was no further gas production with prolonged time of incubation. This result might explain the low correlation coefficients observed with lactulose and inulin compared with sugar beet pulp and wheat starch. However, when similar correlations between incubation time and cumulative gas produced were studied for the data up to 24 h for inulin and lactulose, the coefficient of correlation was dramatically increased to 0.83 and 0.91, respectively.

Similarly, the reduction in pH with incubation time became significant, with a coefficient of 0.80 for inulin and lactulose, when data were calculated for the first 24 h instead of 72 h. In contrast, there was a numeric decrease in pH for inulin and lactulose with incubation time when the data were correlated up to 72 h (Table 2). Concentration of VFA is responsible for lowering pH, but with substrate disappearance approaching zero, production of VFA also slows down. Furthermore, VFA production contributes to gas production (Van Soest, 1994). It is evident (Table 2) that the correlation coefficient for total VFA and cumulative gas produced with inulin and lactulose was lower compared with sugar beet pulp and wheat starch. However, when calculated at 24 h, coefficients increased dramatically for inulin (0.89) and lactulose (0.87). This illustrates that for inulin and lactulose, fermentation until 24 h gives more realistic values than at 72 h.

The time at which 99% of the substrate disappeared was calculated by using the equation described by France et al. (2000). This calculation resulted in 26 h for lactulose, 21 h for inulin, 65 h for wheat starch, and 160 h for sugar beet pulp for the current study. These results are very comparable with time when rate of gas production approached zero (i.e., asymptotic gas production was reached). Table 3 shows that differences in OM disappearance between 24 and 72 h of fermentation were much lower for inulin and lactulose compared with wheat starch and especially sugar beet pulp. Therefore, when asymptotic cumulative gas production was reached, most of the substrate had been fermented, and the fermentable portion had disappeared; this suggests that the fermentation end product profile at the time when asymptotic cumulative gas production is reached is the most appropriate indication of fermentability of a particular substrate in terms of in vitro fermentation end products. This information in relation to end product profiles mentioned in Table 3, suggests that concentrations of end products at 24 h are more appropriate indicators of fermentation end product profile of inulin and lactulose, whereas concentrations of end products at 72 h are more appropriate for wheat starch and sugar beet pulp.

In the present experiment, incubation time influenced (P < 0.01) ammonia, total VFA concentration, and branched-chain VFA proportion. In particular, ammonia concentration and branched-chain VFA proportion, which are typical products of protein fermentation (Macfarlane et al., 1992), increased significantly from 24 to 48 h and increased at 72 h in case of lactulose and inulin. This change in ammonia concentration is also illustrated in Figure 3. Several other studies have shown similar effects of prolonged fermentation (Stefanon et al., 1996; Cone et al., 1997). An experiment reported by Cone et al. (1997) suggested that the rise in ammonia concentration is probably a result of microbial turnover.

View larger version (15K): [in this window] [in a new window]   Figure 3. Ammonia production (mmol/g of OM incubated) during the 72-h fermentation of 4 carbohydrates [inulin (), lactulose (), sugar beet pulp (•), and wheat starch (x)] by fecal inoculum of suckling piglets. The interaction between substrate and incubation time had a significant effect (P < 0.001; SE = 0.11) on ammonia production over time.

View larger version (18K): [in this window] [in a new window]   Figure 4. Lactate acid production (mmol/g of OM incubated) during the 24-h fermentation of 4 carbohydrates [inulin (), lactulose (), sugar beet pulp (•), and wheat starch (x)] by fecal inoculum of suckling piglets. The interaction between substrate and incubation time had a significant effect (P < 0.001; SE = 0.45) on lactate acid production over time.

	Footnotes

 
¹ Supported by the European Union grant "Healthy Pigut" (QLK-LT2000-00522). The authors thank M. Booij, J. Muijlaert, D. Bongers, and H. Boer of the Animal Nutrition Group for their assistance with the laboratory analyses.

² Corresponding author: a.awati{at}massey.ac.nz

Received for publication January 20, 2005. Accepted for publication December 15, 2005.

	LITERATURE CITED

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