Cecum, and Colon of the Pig

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1989, p /89/ $02.00/0 Copyright 1989, American Society for Microbiology Vol. 55, No. 10 In Vitro Total-Gas, CH4, H2, Volatile Fatty Acid, and Lactate Kinetics Studies on Luminal Contents from the Small Intestine, Cecum, and Colon of the Pig JOSEPH A. ROBINSON,* WALTER J. SMOLENSKI, MARVIN L. OGILVIE, AND JAMES P. PETERS Microbiology and Nutrition Research, The Upjohn Company, Kalamazoo, Michigan Received 13 February 1989/Accepted 6 July 1989 Two experiments were conducted to assess differences in fermentative activities of digesta obtained from various regions of the pig gastrointestinal tract. In experiment 1, the contents of small intestines, ceca, and colons of 110-kg pigs were collected, diluted twofold, and incubated for 2 h at 37 C. In experiment 2, colonic samples from kg pigs were similarly treated, except that the incubation period was 5 h. Total gas (gas pressure), CH4, H2, lactate, formate, acetate, propionate, butyrate, valerate, and isovalerate were measured in experiment 1. Only the gas variables were measured in experiment 2. Statistically significant differences (P > 0.05) were not observed among the gas production rate estimates across the small-intestinal, cecal, and colonic regions in experiment 1. Furthermore, all the small-intestinal samples and half the cecal samples assayed in experiment 1 were nonmethanogenic. The mean methanogenic and total-gas production rate estimates for the colonic samples in experiment 1 were ml g of wet contents-' h-' and 1.7 ml of total gas g of wet contents-' h'-, respectively. No differences in the methanogenic rate estimates were detected between the proximal, middle, and distal thirds of the pig colons (P > 0.05). The volatile fatty acid and lactate molar percentages measured in experiment 1 were consistent with previously published observations. Hydrogen accumulated to the greatest extent (7,uM on average) in the in vitro incubations of small-intestinal contents, whereas the H2 concentrations ranged from 0.5 to 1,uM for the incubated cecal and colonic samples in experiment 1. In experiment 2, the variability of the cumulative gas production rate estimates was lower (coefficient of variation, 24%) than that observed for the CH4 production rates (coefficient of variation, 58%). Average rates for total gas production and methanogenesis in experiment 2 were 0.4 ml and ml g of wet contents-' h-, respectively. There was a highly significant (P = ) inverse correlation (r = -0.8) between the average micromolar H2 concentration and the rate of methanogenesis, suggesting that H2 is important methanogenic precursor in porcine colonic samples incubated in vitro. Investigators have indirectly shown, by using respirometry, that methanogenesis occurs in the lower gastrointestinal (GI) tract of the pig (7). Methanogenesis accounts for little of the loss in digestible energy to the pig (7), in contrast to the ruminant (38). Previous work has focused on identifying bacterial species in samples of pig gut contents (1, 20, 22, 25, 26, 30, 39, 40) and feces (21, 31). Few investigators have reported on the numbers of methanogens in the GI tract of the pig (5, 36), and data on methanogenic activities of samples taken from different regions of the GI tract are lacking. No information exists about the relative rates of methanogenesis in different regions of the lower GI tract of the pig. This study was undertaken primarily to generate information on in vitro methanogenic and hydrogenogenic activities of samples of porcine small-intestinal, cecal, and colonic contents. The variation in methanogenic rates for samples collected from the colons of pigs was of specific interest. Interest in the variation in colonic methanogenic activity of incubated pig gut contents is prompted by two observations. First, the GI tract of the pig is similar to that of humans (9, 19, 24), and second, the number of cultivable methanogens in human feces is highly variable (20). Assuming that the microbial community in the lower GI tract of the pig is functionally similar to that in the human lower GI tract, the expectation was that methanogenesis in the colon of the * Corresponding author pig would vary considerably from one pig to the next, as is true for humans (4). This report summarizes data from two experiments. The objective of experiment 1 was to compare in vitro fermentative activities of small-intestinal, cecal, and colonic contents obtained from four pigs. Total gas (pressure), CH4, H2, lactate, formate, acetate, propionate, butyrate, valerate, and isolvalerate were measured in experiment 1. Organic acid concentrations in the pig GI tract have been reported (2, 11, 13-15, 23, 37, 41). The organic acid measurements were made in this study to complement the measurements of total-gas, CH4, and H2 production. In experiment 2, digesta samples from the colons of 16 pigs were incubated in vitro; only the gas variables were monitored. The objectives of experiment 2 were to (i) assess the variation in the production of CH4 and H2 by 16 samples of pig colonic contents and (ii) test the significance of the correlation between CH4 and H2 production. MATERIALS AND METHODS Animals and diet. In both experiments, equal numbers of male and female crossbred (Yorkshire, Hampshire, and Duroc) pigs were used. The 4 pigs in experiment 1 weighed 107 to 118 kg each, and the 16 pigs used in experiment 2 weighed 80 to 90 kg each. All pigs were allowed to consume a corn diet (Table 1) ad libitum and were given free access to water. Sampling protocols. In experiment 1, the pigs were anesthetized with ketamine hydrochloride (Warner-Lambert Co., an

2 VOL. 55, 1989 KINETICS OF FERMENTATION IN PIG GUT LUMINAL CONTENTS 2461 TABLE 1. Composition of diet fed to the pigs in both experimentsa Ingredient Amtb Corn (coarse ground) Soybean meal Dicalcium phosphate Limestone (calcium) Salt (iodized) Dyna K (KCI)C Swine Premix 5sc (vitamins and trace minerals) a Analysis of the diet showed the following composition: protein, 16.1%; fat, 3.1%; fiber, 2.8%; calcium, 0.68%; phosphorus, 0.62%; potassium, 0.69%o; magnesium, 0.17%; lysine, 0.74%; methionine and cystine, 0.54%; gross energy, 3,098 kcal/kg (12,962 kj/kg). Feed analysis was done by the swine extension service at Michigan State University, East Lansing. b Percentage on an as-fed basis. c From Carl S. Akey, Inc., Lewisburg, Ohio. Morris Plains, N.J.) and Rompun (Miles Laboratories, Inc., Shawnee, Kans). A midline incision was made along the ventral surface of the abdomen, exposing the lower GI tract (for our purposes, the lower GI tract is defined as the region from the proximal small intestine to the distal colon). String was used to tie off the various regions to minimize movement of digesta. Once this in situ ligation was completed, the entire lower GI tract from the proximal small intestine to the distal colon was removed. Immediately after this step, the four pigs were exsanguinated. The small intestine, cecum, and colon were separated from one another, and the length of each region was measured. The small intestine was cut into two equal lengths, and the contents from each region were collected in separate 2-liter Erlenmeyer flasks being sparged with anaerobic CO2. The cecum was left intact, its length was measured, and the contents were emptied into another C02-containing flask. The colon was divided into three equal lengths, and the contents of each were collected as described above. In addition to the lengths, the empty weights of each region of the gut (small intestine, large intestine, and cecum) were recorded. Before being weighed, the gut sections were washed in 0.9% physiological saline and blotted dry. In experiment 2, colonic contents from 16 pigs were collected. In this case, the entire gut was removed after the pigs were sacrificed by using a captive bolt, and the contents from the proximal colon (1 to 2 m) were transferred into separate 2-liter flasks being sparged with anaerobic CO2. Incubation protocols. Samples were taken to the laboratory for processing within 5 min of collection and diluted 1:1 (by weight) with an anaerobic buffer (16). We modified the buffer to exclude both major and minor volatile fatty acids. A given, diluted sample was passed through two layers of cheesecloth into a 500-ml Erlenmeyer flask being sparged with anaerobic CO2. A 45-ml sample of the filtered sample was anaerobically transferred to a 100-ml serum bottle, and the bottle was sealed with a rubber stopper and placed in a 37 C rotary shaking water bath set at 250 rpm. The incubation periods were 2 and 5 h for experiments 1 and 2, respectively. Duplicate bottles were run in experiment 2, unless the sample volume was prohibitively small. Duplicate bottles were also filled in experiment 1, but one set was frozen immediately for zero-time measurements of the organic acids. Total-gas, H2, and CH4 measurements. The total-gas volume was measured by using a pressure transducer (model PX GV; Omega Engineering, Inc., Stamford, Conn.). The signal from the transducer was read as voltage by using a multimeter. Gas was expressed as cumulative milliliters produced during the incubation, per volume of undiluted contents (approximately 23 g). CH4 and H2 in headspace samples were analyzed by gas chromatography. CH4 was separated from the rest of the gases in an injected sample by using a Poropak Q column (100/120 mesh; 6 ft [1.8 m] by 1/8 in. [0.3 cm] [outer diameter]). CH4 in the gas sample was detected by using a flame ionization detector (Varian, Palo Alto, Calif.). H2 was separated on a molecular sieve 5A column (45/60 mesh; 3 ft [0.9 m] by 1/4 in. [0.3 cm] [outer diameter]) and detected by using a mercury reduction gas analyzer (Trace Analytical, Menlo Park, Calif.). Chromatographic-grade helium (Linde Division, Union Carbide Co., Somerset, N.J.) was used as the carrier and was set at 30 and 60 ml min-1 for CH4 and H2 analyses, respectively. The H2 and CH4 peaks were quantitated by using a computing integrator (HP-3393A; Hewlett- Packard Co., Avondale, Pa.). Methane was expressed as cumulative milliliters (dependent on the gas pressure measurement) per gram of undiluted contents (23 g) per incubation. H2 was expressed as the concentration of this gas dissolved in the liquid phase. A solubility coefficient (42) for H2 dissolved in water at 37 C was used to calculate the dissolved concentration of H2 in equilibrium with the ppm H2 values measured in the headspace. The contribution of dissolved CH4 to cumulative CH4 production was ignored, since it would have accounted for about 2% of the CH4 in the entire sealed bottle, given the solubility of CH4 (42) and the ratio of gaseous to liquid phase volumes used in the incubations (10). The H2 concentrations present in the in vitro incubations of diluted small-intestinal samples produced signals exceeding 1 V, which is the maximum voltage for the integrator used. The measured H2 concentrations for these samples are flagged in the Results section as being at least as high as the given values. Formate, lactate, acetate, propionate, butyrate, valerate, and isovalerate measurements. Samples from the zero-time and incubated bottles from experiment 1 were stored at -20 C until analysis. The samples were thawed and processed on ice in preparation for quantitation of the organic acids by high-performance ion chromatography (29). Samples (1 ml) were removed from each incubation bottle, diluted in 9 ml of 50 mm HCI, and centrifuged at 14,000 x g for 4 min. Each clarified supernatant was pipetted into a plastic vial, stoppered, and stored at 4 C. Volatile and nonvolatile fatty acids in the thawed, processed samples were analyzed by previously described procedures (Dionex Ion Chromatography Cookbook; Dionex, Sunnyvale, Calif., 1987). Regression analyses. Regression analysis was used to generate a single summary for each total-gas and CH4 production curve. The summary of interest was the initial rate of total-gas or CH4 production. The first step was to decide which equation (of the three tried) best described the data. The total gas and CH4 progress curves were each fit to a straight line and to two nonlinear models. These models were then used to estimate the initial total-gas and CH4 production rates. Data were fit to a straight line (total gas or CH4 = k1 + k2 x time) (model 1) by using PROC REG from SAS (34). The nonlinear models used were as follows: total gas or CH4 k3[1 = - exp (-k4 x time)] (model 2) and total gas or CH4 = k3[1 - exp (-k4 x time)] + k5 (model 3). Both of these nonlinear models are first-order appearance equations, but

3 2462 ROBINSON ET AL. the second accounts for the possibility that a given production curve may not have its origin at zero. The nonlinear models were fit to the total-gas and CH4 production curves by using PROC SYSNLIN from SAS (33). The best of the three models was defined as the one which gave a residual sum-of-squares that was at least 25% lower than the residual sums-of-squares obtained for the other two equations. A similar approach has been suggested by Mannervik (17), given the limitations of the F test for model discrimination (3, 17, 27). Summary variables for total-gas and CH4. As indicated above, a single summary was defined for each production curve in the form of a rate estimate. Initial velocity estimates for total gas and CH4 production were estimated by using the best of the three models fit to a given progress curve. For the linear model, the initial velocity estimate equals the slope (k2). For either nonlinear model, the initial velocity estimate equals the instantaneous slope of the curve at time zero k3k4. Notice that the presence of a nonzero intercept does not affect the initial velocity estimate for gas or CH4 when first-order kinetics is obeyed. Summary variables for H2 production. Dissimilarities among the kinetic patterns for H2 accumulation in the incubations necessitated an approach different from that used to summarize the gas and CH4 progress curves. The mean, median, and maximum dissolved H2 concentrations were computed by using PROC UNIVARIATE from SAS (32) for each of the incubated bottles in the two experiments. These three summaries were used in the correlational and univariate analyses. As noted above, the H2 concentrations in the headspaces above the incubated luminal contents obtained from the small intestine often produced voltages that exceeded the maximum input voltage for the integrator (1 V). Thus, the resulting estimates of the minimum, median, and maximum dissolved H2 concentrations are too low, being at least as high as the stated values. These values are flagged in Table 3, in which the H2 values for the various lower gut regions of the pig are summarized. Organic acid summary variables. The individual volatile fatty acids and lactate (nonvolatile fatty acid) were expressed as molar percentages, for both the zero-time and incubated samples. The total was defined as the sum of the millimolar concentrations of formate, acetate, propionate, butyrate, isovalerate, valerate, and lactate for a given region of the pig GI tract. The total volatile plus nonvolatile fatty acid content was statistically analyzed along with the molar percentage estimates. Statistical comparisons and correlations. For experiment 1, values obtained for each variable (e.g., lactate molar percentage, CH4 production rate) were analyzed by using the Repeated Option of PROC GLM from SAS (34). For these analyses, the region of the lower gut of the pig represents the repeated factor. For experiment 2, the total gas and CH4 production rate estimates, plus the H2 production summary variables (minimum, median, and maximum H2 concentrations), were summarized overall by using PROC UNIVARIATE from SAS (32). The Shapiro-Wilk test (32) was used to assess whether the total-gas production summary variables estimated in experiment 2 were other than normally (Gaussian) distributed. Quantile-Quantile plots (6) were used to corroborate the results of the Shapiro-Wilk tests. Correlational analyses were done by using PROC CORR from SAS (34) to determine the nature and significance of correlations among the rate estimates for CH4 and total-gas production versus the minimum, median, and maximum H2 concentration estimates obtained in experiment 2. RESULTS APPL. ENVIRON. MICROBIOL. Wet weights of luminal contents from the various regions (experiment 1). The wet weights of the cecal contents ranged from 0.33 to 0.39 kg, whereas the wet weights of the luminal contents from the first and second small-intestinal regions ranged from 0.20 to 0.36 kg and from 0.10 to 0.62 kg, respectively. The wet weights of the luminal contents from the proximal, middle, and distal thirds of the colons of the four pigs ranged between 0.49 and 0.64, 0.36 and 0.38, and 0.10 and 0.11 kg, respectively. Total-gas, CH4, and H2 production by the small-intestinal, cecal, and colonic pig gut samples (experiment 1). Except for three of the incubations of small-intestinal contents, all incubations in which total gas production was measured in experiment 1 were best described by a first-order kinetic model with a nonzero intercept. Again, the criterion used was that for a given model to be judged superior, it had to yield a residual sum-of-squares 25% lower than that achieved for the other two models. The CH4 production progress curves for experiment 1 were accurately described, in general, by a linear model. CH4 was not produced by the small-intestinal and half of the cecal samples over the 2-h incubation period. The pattern of H2 accumulation was dependent on the region from which the luminal contents were collected (Fig. 1). The H2 concentrations plotted for the small-intestinal regions are at least as high as the plotted values beyond about 3,uM, since H2 partial pressures in equilibrium with more than 3,uM produced output voltages that could not be reliably quantitated. Excluding the samples obtained from the two halves of the small intestine, the kinetic patterns of H2 accumulation were similar (Fig. 1). Dependence of total-gas and CH4 production rate estimates on lower gut region of the pig (experiment 1). As stated in Materials and Methods, the purpose of the regression analyses was to determine the best model for estimating the initial rate of total-gas and CH4 production for a given incubated sample. Model 3 (first order with intercept) was used to estimate the initial rate of total gas production for the samples incubated in experiment 1. The r2 values for the fits of model 3 to the cumulative gas progress curves all exceeded (Table 2). An estimate of the total-gas production rate for a given sample was obtained by multiplying the estimates of k3 and k4. No significant differences (P > 0.05) were detected among the initial total-gas production rate estimates obtained for samples from the various regions (Table 2). The linear model (model 1) was generally superior to the two nonlinear models (models 2 and 3) and therefore was used to estimate the initial CH4 production rates for the samples incubated in experiment 1. Coefficients of determination for fits of the CH4 accumulation progress curves to the linear model exceeded 0.93 (Table 2). The region of the lower pig gut did have a statistically significant effect on the estimate of the CH4 production rate, largely because zero production rate estimates were obtained for all of the smallintestinal samples and for two of the four sets of incubated cecal samples (Table 2). Significant differences (P > 0.05) in the CH4 production rate estimates for the three colonic regions were not observed (Table 2). The average CH4 production rate for the cecal samples obtained from the four pigs was 30-fold lower than the mean CH4 production rate

4 VOL. 55, 1989 KINETICS OF FERMENTATION IN PIG GUT LUMINAL CONTENTS c &12 0 *0 2 I 6 A nō REGION=smallinl j c o12 0 2IA6 REGION=smollin2 wooo- V L I *0 >5 32 n,6-5. REGION=cecum..O o I 0, 10 Ti a, REGION=colonl L. la O REGION=colon2 C 0 L >5 2z A REGION=colon3 FIG. 1. Calculated dissolved H2 concentrations for the incubated samples collected from the two small-intestinal regions, the cecum, and the three colonic regions (experiment 1). The calculated dissolved H2 concentrations are based on headspace measurements of the incubated serum bottles. The H2 concentrations shown for the small-intestinal regions sampled are at least as high as the plotted values, since the maximum input voltage for the integrator was exceeded. Four curves are shown for each of the six panels; one curve is shown per sampled pig. estimated for the samples collected from the last one-third of the sampled colons (P < 0.05 [Table 2]). Influence of gut region on the H2 summary variables (experiment 1). For the reason cited in Materials and Methods, TABLE 2. Average initial rate estimates for total-gas and CH4 production obtained for samples from the porcine lower GI tract in experiment 1 Region Total-gas production CH4 production Ratea r2 rangeb Ratea r2 rangeb Small intestine Sample x x Sample x x Cecum 2.31x x Colon Sample x x,y Sample x x,y Sample x y a Initial rate estimates for gas production were calculated by using model 3 (first order with intercept); initial rate estimates for CH4 were obtained from the slope of model 1 (linear). Units for the rates are milliliters of gas or CH4 per gram of wet contents per hour. Means followed by different letters differ at the P = 0.05 level of significance. b Range of coefficients of determination for the best-fit equations used to calculate the gas and CH4 production rates; coefficients are not given for CH4 production by the small-intestinal and two of the cecal samples, since CH4 production rates for these samples were zero e51)00 15 the H2 concentrations for the small-intestinal regions exceed the stated micromolar values (Table 3). Owing to this uncertainty, the H2 summary variables for experiment 1 were not analyzed statistically. Empirically, the highest levels of H2 were measured in the samples of incubated small-intestinal contents (Table 3). Molar percentages of the organic acids in lower-gut contents (experiment 1). The highest molar percentages of lactate and formate were found for the two small-intestinal sections (Table 4). The molar percentages of lactate and formate in the small-intestinal regions were not different (P > 0.05 [Table 4]). The molar percentage of acetate in the first region of the small intestine was 18%, increasing to 70% in the colon (Table 4). No differences (P > 0.05) in the molar percentage of acetate were detected across the cecum and the three colonic regions (Table 4). The molar percentage of propionate in the small-intestinal regions was less than 0.05%, whereas the molar percentage of propionate in the cecum and first two regions of the colon was about 20% (Table 4). The molar percentages of propionate for the cecum (24%) and the third colonic region (19%) were different (P < 0.05 [Table 4]), although the numerical difference is probably not meaningful. The molar percentage of butyrate increased (P < 0.05) from less than 0.05% for the first small-intestinal region to IF n w 0

5 2464 ROBINSON ET AL. TABLE 3. Region H2 summary variables for the incubations of lower GI tract contents incubated in experiment 1 H2 concna Maximum Minimum Median Small intestine Sample *b * Sample * 4.34* 8.95* Cecum Colon Sample Sample Sample a Units for all four summary variables are micromolar dissolved (calculated, based on headspace measurements). Values given are means across the four pigs sampled, per region. b Asterisks indicate that the concentration is at least as great as the value in the table. 7% in the colonic regions (Table 4). Differences were observed between the molar percentages estimated for the second small-intestinal, the cecal, and the colonic regions (Table 4). The molar percentages of isovalerate and valerate were less than 2.0% for all of the sampled regions. The molar percentage of isovalerate was greater than 0.05% for only the third colonic region (Table 4). The molar percentage of valerate was less than 0.05% in the small-intestinal and cecal samples (Table 4); it averaged 1.5% in the three colonic regions, with no differences across the regions (P > 0.05 [Table 4]). Total millimolar concentrations of volatile and nonvolatile fatty acids did not differ across the cecum and the first two colonic regions (mean concentration, 150 mm). This value was significantly (P < 0.05) different, however, from the millimolar concentrations measured for the first (20 mm) and second (80 mm) small-intestinal regions and the concentration (130 mm) observed for the third colonic region. The last three values all differed from one another at the 5% level of significance. APPL. ENVIRON. MICROBIOL. Molar percentages of the organic acids after the 2-h incubation period (experiment 1). Lactate molar percentages declined in the cecal and colonic samples during the 2-h incubation, but the differences among the regions paralleled the differences observed for the zero-time samples (Table 4). The pattern of high formate and lactate molar percentages in the small intestine, with low molar percentages of the volatile fatty acids excluding acetate, was still evident after 2 h of incubation (Table 4). Except for lactate, butyrate, and isovalerate, differences in the molar percentages of the organic acids were not observed across the cecal and three colonic regions (P > 0.05 [Table 4]). For the incubated samples, the sum of volatile plus nonvolatile fatty acids averaged across the three colonic regions was 220 mm. This value is different (P < 0.05) from the total concentrations measured for the first (66 mm) and second (150 mm) small-intestinal regions and the cecum (190 mm). Furthermore, the total millimolar concentrations measured in these three regions all differed (P < 0.05) from one another. Total-gas and CH4 production by replicate colonic samples (experiment 2). The major objective of experiment 2 was to assess the variability in total-gas, CH4, and hydrogen production by samples of pig colonic contents obtained from 16 pigs. The correlation between the H2 concentrations and the methanogenic rate estimates was also of interest. If H2 is a major precursor of methanogenesis in pig gut samples incubated in vitro, a negative correlation should exist between the initial rate of methanogenesis and the median H2 concentration, assuming that gas phase measurements of H2 are meaningful relative to the liquid phase concentrations of H2. For total-gas production, the first-order model with a nonzero intercept (model 3) best described the progress curves for all 16 samples. Model 3 was also superior to models 1 and 2 for 15 of the 16 CH4 progress curves. The fits of model 3 to the entire set of 32 progress curves were good; the r2 values for the 32 nonlinear regressions all exceeded TABLE 4. Region and time Volatile and nonvolatile fatty acid molar percentages for the porcine lower GI tract samples collected in experiment 1, before and after a 2-h incubation Molar % of: Lactate Formate Acetate Propionate Butyrate Isovalerate Valerate Before incubation Small intestine Sample 1 68x 14x,y 18x 0.Ox 0.0w 0.Ox 0.Ox Sample 2 29x 27y 43y 0.Ox 1.4x 0.Ox 0.Ox Cecum 0.9y 0.Ox 70z 24y 4.8y 0.Ox 0.Ox Colon Sample 1 1.7y 0.Ox 70z 20y,z 6.lz 0.Ox 1.4y Sample 2 2.6y 0.Ox 69z 20y,z 6.5z 0.Ox 1.5y Sample 3 4.6y 0.Ox 67z 19z 6.6z LOy 1.6y After incubation Small intestine Sample 1 51x 20x 28x 0.7x 0.Ox 0.Ox 0.Ox Sample 2 30x 19x 46y 0.lx 3.1y 0.Ox 0.Ox Cecum 0.Oy 0.Oy 66z 26y 6.5z 0.3x 0.9y Colon Sample 1 0.3z 0.Oy 60z 29y 7.9z 0.8y 2.2y Sample 2 0.4z 0.Oy 61z 27y 8.lz 1.1y 2.2y Sample 3 0.7z 0.Oy 62z 25y 7.9z 2.Oy 2.4y a Mean values for three pigs (samples for the fourth were lost). Means for a given variable followed by different letters are significantly different (P < 0.05). Total amounts are given in the Results section.

6 VOL. 55, 1989 KINETICS OF FERMENTATION IN PIG GUT LUMINAL CONTENTS 2465 TABLE 5. Summaries of the total-gas production variables measured in experiment 2 Summarya Mean + SD CV (%) Range Gas rate ± CH4 rate ± H2 concn Maximum ± Median ± Minimum a Units for total-gas and CH4 production rate estimates are cumulative milliliters of gas or CH4 per gram of wet undiluted contents per hour. Both sets of rate estimates were computed by using model 3. Units for the three H2 summary variables are micromolar dissolved (calculated based on headspace measurements). The relative variation in methane production (coefficient of variation, 58%) was greater than that observed for cumulative gas production (coefficient of variation, 24%) by the 16 incubated samples of colonic contents (Table 5). The average total-gas production rate was 0.43 ml g of undiluted wet contents-1 h-1, with a coefficient of variation of 24% (Table 5). The sample coefficient of variation for the initial rate of CH4 production was 58%, with a mean rate of ml g of undiluted wet contents-' h-1. Probability levels for tests of the null hypothesis of normality for the total-gas and CH4 production rate estimates obtained were 0.01 and 0.58, respectively. The null hypothesis of normality for the total-gas production rate estimates was rejected because of the influence of the lowest gas production rate estimate. Otherwise, the two batches of rate estimates behaved similarly to that expected for Gaussiandistributed data. Quantile-quantile plots (not shown) of the total-gas and CH4 rate estimates were linear, supporting the conclusion that the two sets of rate estimates were normally distributed. H2 production by colonic samples (experiment 2). The H2 accumulation curves obtained for the 16 replicates (Fig. 2) exhibited different kinetic patterns, as noted in experiment 1 (Fig. 1). This was in contrast to the total-gas and CH4 progress curves, which all behaved similarly, as evidenced by the quality of the fits to the best model used to summarize these data. Since the diversity of the accumulation patterns for H2 could not all be described by using a single regression model, three simple summaries of each H2 accumulation curve were used in the correlational and univariate statistical analyses. As in experiment 1, these summaries were the minimum, median, and maximum calculated liquid phase H2 concentrations. The average maximum, median, and minimum micromolar H2 concentrations for the 16 incubated samples were 0.82, 0.66, and 0.44,uM, respectively (Table 5). The coefficients of variation for the three H2 summary variables ranged from 50 to 59% (Table 5). Correlation between the H2 concentration and the initial methanogenic rate (experiment 2). A significant negative correlation (r = ; P = 0.003) was detected between the initial rate of methanogenesis and the median micromolar H2 concentration measured in the 16 incubations. The magnitude of this correlation (r = ; P = ) was substantially improved by ignoring the two extreme data pairs (Fig. 3). DISCUSSION Our results indicate that the colon is the site of most of the CH4-producing activity in the lower GI tract of the pig. C0) G_ 0 L > -u0 uco) C] ' / FIG. 2. Calculated dissolved H2 concentrations for the 16 replicate colonic samples incubated in experiment 2. The datum points are not plotted as symbols to avoid clutter. They (five points per plot generally) are shown connected by straight-line segments. Methanogenesis in the cecum of the pig is highly variable and less important quantitatively than CH4 production by colonic samples. The extent of methanogenesis in the cecum was highly variable and more than 10-fold lower than that observed for similarly treated samples of colonic contents. These results are consistent with recently published findings on numbers of methanogens in the cecum and colon of the growing-finishing pig (5). Although dissolved CH4 was found in the small-intestinal samples, CH4 production by incubated small-intestinal contents was not observed in our study. Methanogenesis may be more quantitatively important in the ceca of pigs fed diets high in fiber. It is known that diet does have an effect on the fermentation pattern in the lower gut of the pig (14, 37). In addition to assessing the variation in CH4-producing activities of pig GI tract digesta samples, the second experiment was run to generate data on the sign and strength of the correlation between the rate of methanogenesis and the average concentration of H2 measured in vitro. A negative correlation should exist between the rate of CH4 production and the average H2 concentration assuming that (i) H2 is a quantitatively important methanogenic precursor in incubated samples of colonic pig gut contents, (ii) H2 production and consumption are at or near steady state, and (iii) the headspace and aqueous H2 concentrations are at or near equilibrium. If any of or all of these conditions do not hold, a weak correlation would be expected between the average H2 concentration and the initial rate of methanogenesis. Excluding two pairs of extreme observations, the inverse

7 2466 ROBINSON ET AL c 0. 0) :> X o o ml Methane/g/h FIG. 3. CH4 rate versus the median concentration of H2 (calculated micromolar liquid phase concentration). The CH4 production is expressed in milliliters of total CH4 per gram of wet contents per hour. correlation between the median micromolar H2 concentration and the initial rate of methanogenesis was high (r = 0.95). Hence, the data collected in the second experiment do not refute the conclusion that H2 is an important methanogenic precursor for incubating samples of colonic contents. The amount of H2 measured in the in vitro incubations of small-intestinal contents exceeded that produced in the incubations of the cecal and colonic contents. Similarly, the molar percentages of both formate and lactate were highest for the small-intestinal samples. For the samples from the ceca and colons, the calculated dissolved H2 content is similar to, albeit slightly lower than, the reported ruminal H2 concentration (12, 28, 35). Furthermore, the molar percentages of formate and lactate, before and after a 2-h incubation, were low (or nondetectable) for the gut contents collected from the ceca and colons of the pigs used. The GI tract of the pig is presumed to be functionally similar to the human GI tract (9, 18, 19, 24). Furthermore, the numbers of methanogens in humans span 10 orders of magnitude (20). Given these observations, it was anticipated that methanogenic activity in the lower pig gut would be highly variable. However, while the variation of CH4-producing activity in colonic samples was greater than that seen for total-gas production, the variation of the CH4 rate estimates was not as great as expected. Therefore, although the pig lower gut shares some characteristics with the human lower gut, the two gut systems differ substantially with respect to the variation in apparent methanogenic activity. In particular, the highly skewed (non-gaussian) frequency distributions observed for methanogenic activity in humans (4) contrasts with the apparent symmetrically distributed CH4 production rates observed by us for pig colonic samples. Whether this situation would hold for pigs fed highfiber rather than high-corn diets is unknown. The major focus of this investigation was on total gas, CH4, and H2 production by the contents of selected regions of the pig GI tract. The measurements of the organic acids were done to check the relevancy of the in vitro approach used. The results we obtained for the volatile fatty acid and lactate molar percentages are consistent with previous findings for pigs and for samples from the lower GI tracts of ruminants (8). These findings support the significance of the gas production measurements. The estimates of CH4 production for the pig gut samples obtained in this study were determined by using filtered, twofold-diluted material incubated under ideal conditions. Notwithstanding this artificiality, it was of interest to assess the accuracy of our CH4 production estimates relative to whole-animal CH4 excretion rates previously determined for similar-sized pigs. Chistensen and Thorbek (7) reported data on CH4 excretion by pigs fed a diet similar to the one used in this study. Their data indicate that an 85-kg pig should excrete 5 to 7 liters of CH4 day-l. From the present study, multiplying the mean CH4 production rate of 1.2 liters of CH4 kg of wet contents-1 day-1 (experiment 2) by the average weight of colonic contents (1.0 kg) would, by extrapolation, lead to a CH4 excretion rate of 1.2 liters of CH4 85-kg pig-1 day-1. This rate estimate is clearly much lower than the actual CH4 excretion rate expected for an 85-kg pig. Therefore, although our estimates of methanogeneic activity are useful for comparative purposes, they underestimate the CH4 excretion rate expected for the whole animal. This result is not surprising, given that after the lower-gut samples were diluted twofold, they were passed through two layers of cheesecloth to remove large particles. Presumably, this treatment resulted in a loss of methanogens (as well as organisms supplying the methanogens with H2 and formate) that were present in the samples of colonic contents at the time of collection. The magnitude of the loss in microbial activity due to filtration through the cheesecloth is APPL. ENVIRON. MICROBIOL. unknown. In summary, total-gas production did not differ across the pig small intestine, cecum, and colon in this study. In contrast, considerable differences were observed for rates of CH4 production across regions of the porcine lower GI tract. The variation in methanogenesis was high for the colonic samples but not as high as expected, given similarities between the GI tracts of humans and pigs. Incubations of small-intestinal contents were characterized by high H2, formate, and lactate levels, in contrast to the cecal and colonic samples. Similarities between the molar percentages of organic acids reported in this study, relative to previous findings, support the relevancy of the inferences drawn from the gas production measurements performed. ACKNOWLEDGMENTS We thank Robert Bell and Richard Y. W. Shen for help in sample collection. Thanks also go to Karen Barsuhn for the organic acid analyses. LITERATURE CITED 1. Allison, M. J., I. M. Robinson, J. A. Bucklin, and G. D. Booth Comparison of bacterial populations of the pig cecum and colon based upon enumeration with specific energy sources. Appl. Environ. Microbiol. 37: Argenzio, R. A., and M. 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