Influence of High-Fiber Diet on Bacterial Populations in Gastrointestinal Tracts of Obese- and Lean-Genotype Pigs

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1982, p. 17-112 99-224/82/717-6$2./ Vol. 44, No. 1 Influence of High-Fiber Diet on Bacterial Populations in Gastrointestinal Tracts of Obese- and Lean-Genotype Pigs V. H. VAREL,* W. G. POND, J. C. PEKAS, AND J. T. YEN Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, Nebraska 68933 Received 2 November 1981/Accepted 23 March 1982 Bacterial populations from gastrointestinal tracts of genetically lean and obese pigs fed a low- or high-fiber diet ( or 5% alfalfa meal, respectively) were enumerated with rumen fluid media and specific energy sources. Total culture counts in rectal samples declined 56 (P >.5) and 63% (P <.5) in lean and obese animals, respectively, 3 weeks after feeding the high-fiber diet. After 8 weeks, culture counts had risen and were similar to those obtained before alfalfa was fed ( week). At slaughter, 12 to 17 weeks after feeding the high-fiber diet, total counts from rectal samples of lean pigs continued to rise and were 13% greater than the -week counts, whereas counts from obese animals declined 37% (P >.5). The number of cellulolytic bacteria in rectal samples of lean-genotype pigs fed the high-fiber diet increased 8 and 71% from to 3 weeks and 3 to 8 weeks, respectively. This overall increase from to 8 weeks in lean pigs was significant (P <.5); however, these increases were not seen in obese pigs. These data suggest that the microflora is initially suppressed when exposed to a high-fiber diet and that later some adaptation takes place, apparently more so in lean than in obese pigs. When specific energy sources were used to delineate the distribution of different bacterial populations in the cecum, colon, and rectum, trends could be detected between high- and low-fiber diets. These data also support the concept that bacterial populations from different sites in the large bowel differ. Information on the utilization of dietary fiber for energy by nonruminant species is limited. There is evidence for individual variation among humans (3, 8; J. L. Jeraci et al., Abstr. Am. Soc. Anim. Sci., abstr. no. 245, 198) and pigs (1, 15) in their ability to utilize dietary fiber. Betain et al. (3) found cellulose-digesting bacteria in fecal samples from only one of five human subjects, and the rate of cellulose degradation of the Bacteroides sp. isolated was lower than that of rumen isolates. These results suggested that diet or genetic influences were involved in the ability to harbor active cellulolytic bacteria. In contrast, Slavin et al. (2 found no significant variation in fiber digestibility among seven human subjects; however, fiber digestion decreased from 7 to 23% when the subjects were placed on a high-fiber diet. Bornside ( concluded that the major groups of bacteria and their concentration in gastrointestinal tracts of humans are not altered by dietary fiber. This conclusion is supported by the work of Salyers et al. (23), who reported that anaerobic bacteria from the human colon have inducible rather than constitutive enzymes, and therefore, the same microbial flora could have very different overall metabolic activities, depending on the diet of the host. 17 The present study determined the effect of low- and high-fiber diets on the bacterial population in the feces and intestinal tracts of obeseand lean-genotype pigs. Thus, our experiments were designed to enumerate the bacterial populations from genetically lean and obese pigs fed a low- or high-fiber diet ( and 5% alfalfa meal, respectively). Total viable cells and cellulolytic bacteria from feces were enumerated at, 3, and 8 weeks after pigs were fed the high-fiber diet to determine whether counts were altered. At slaughter, bacterial populations were compared from the cecum, colon, and rectum, using specific energy sources (1, 7). MATERIALS AND METHODS Animals and sampling. Six obese-genotype and six lean-genotype crossbred Duroc x Yorkshire females derived from inter se matings of pigs from populations selected over multiple generations for low and high backfat (12) were used in this study. Pigs weighing approximately 6 kg each were randomly assigned to individual pens and fed either a conventional low-fiber or a high-fiber (alfalfa meal) diet. Neither diet contained antibiotics. The low-fiber diet contained 78% ground corn, 18% soybean meal, and.5% limestone, plus vitamins and minerals. This diet was also fed to all pigs before the start of the experiment. The high-fiber Downloaded from http://aem.asm.org/ on July 4, 218 by guest

18 VAREL ET AL. diet contained 5% dehydrated alfalfa meal, 32.5% ground corn, and 14% soybean meal, plus vitamins and minerals. Diets were fed ad libitum until slaughter. All gastrointestinal and rectal samples of fecal material were processed by the methods described by Salanitro et al. (21), except the subsample of material used for fatty acid analysis was centrifuged for 3 instead of 1 min. Rectal samples were cultured before feeding the high-fiber diet to establish baseline data. Thereafter, rectal samples were obtained at 3 and 8 weeks and at slaughter (12 to 17 weeks) after feeding the respective diets. Pigs were fasted (16 to 18 h) before they were slaughtered at approximately 14 kg. The time between slaughter and sampling of the cecum, colon, and rectum was 2 to 3 min. The colon was sampled at the apex of the spiral colon. Cultural procedure and media. A basal medium which contained 3% incubated, clarified rumen fluid was prepared by the procedure of Dehority and Grubb (7) with the modifications of Allison et al. (1). Before incubation, the rumen fluid ph was 7.1, and concentrations of acetate, propionate, and butyrate were 55, 12, and 7 mm, respectively. After the rumen fluid, agar, minerals, and resazurin were incubated for 7 days at 38 C, the ph was unchanged, and concentrations of acetate, propionate, and butyrate were 84, 18, and 12 mm, respectively. Total viable count (TVC) medium consisted of the incubated basal medium (3% rumen fluid, minerals, resazurin, undissolved agar, water) plus the following (in grams per liter): glucose, cellobiose, starch, xylose, maltose, and glycerol, each.3; Trypticase (BBL Microbiology Systems), 2.; and hemin,.125. When substrates were added individually to the basal medium, they were at the following concentrations: glucose, cellobiose, starch, xylose, maltose, or mannitol, each 1 g/liter; Trypticase, 2 g/liter; and sodium lactate, 2 g/liter. The Hungate anaerobic culturing method as described by Bryant (5) was used throughout the study. Sodium carbonate and cysteine hydrochloride were added as sterile anaerobic solutions, and all media were dispensed under CO2. The medium of Bryant et al. (6) was used for estimating most probable numbers (three tubes) of cellulolytic bacteria. Volatile fatty acdds in cecum, colon, and rectal samples were determined by gas chromatography (26). Data were analyzed by least-squares analysis of variance, as described by Harvey (11), and Duncan's multiplerange test (25). RESULTS Total viable counts. Colony counts from rectal samples of genetically lean and obese pigs fed rations containing low or high fiber were compared at various time intervals after feeding the diets (Table 1). Culture counts declined 56 (P >.5) and 63% (P <.5) in lean and obese animals, respectively, 3 weeks after feeding the high-fiber diet. During the same time, culture counts from rectal samples of lean and obese animals fed the low-fiber diet also declined 23 and 41% (P >.5), respectively. We attributed this difference in colony counts between 23 and 56% (33%) and 41 and 63% (22%) to diet. After 8 APPL. ENVIRON. MICROBIOL. weeks on the high-fiber diet, colony counts had returned to initial (-week) levels. At slaughter, 12 to 17 weeks after feeding the high-fiber diet, the total counts from rectal samples of lean pigs continued to rise and were 13% greater (P >.5) than the -week counts, whereas counts from obese animals declined 37% (P >.5). Cellulolytic populations. A comparison of the cellulolytic population from these animals is shown in Table 2. A significant change (P <.5) was observed over an 8-week period in lean animals fed the high-fiber diet, in which the cellulolytic population increased 8 and 71% from to 3 weeks and 3 to 8 weeks, respectively. Only a minimal and nonsignificant (P >.5) increase was observed during this same period in obese animals fed the same diet. Samples for cellulolytic counts were not obtained at slaughter. Volatile fatty acids. A comparison of the concentrations of volatile fatty acids obtained in the cecum, colon, and rectum at slaughter is shown in Table 3. The concentration of propionate found in the colons and rectums of lean animals and in the rectums of obese animals fed the highfiber diet was higher than that found in animals fed the low-fiber diet. This in turn significantly lowered the acetate-propionate ratio (P <.5). Also of interest was the similarity of the acetatepropionate ratios among the ceca, colons, and rectums of animals fed the low-fiber diet and the significantly different ratios (P <.5) among the ceca and colon-rectums of both lean and obese animals fed the high-fiber diet. Specific energy sources. A general trend was observed in both obese- and lean-genotype animals, irrespective of diet, for the percentage of the total viable population metabolizing a specific energy source to decline from the proximal to the distal end of the large bowel (Table. Another trend observed was for the percentage of the total bacterial population using the hydrolytic products of cellulose (glucose and cellobiose) to be higher in the ceca and colons of lean animals fed the high-fiber diet than in those fed the low-fiber diet. This trend was not consistent in obese animals, suggesting that the cellulolytic activity or number of cellulolytic bacteria was less in these pigs. The percentage of the total population metabolizing xylose was 44% greater (P >.5) in the ceca of lean pigs fed the high-fiber diet than in those fed the lowfiber diet. However, this was just the opposite in the rectum, where the percentage of the total population using xylose was approximately 42% more (P >.5) in animals fed the low-fiber diet. Essentially no differences in percentages of total population using xylose were observed between obese animals fed the low-fiber diet and those fed the high-fiber diet, although the declining Downloaded from http://aem.asm.org/ on July 4, 218 by guest

VOL. 44, 1982 BACTERIAL COUNTS FROM PIGS FED HIGH FIBER 19 TABLE 1. Comparison of mean colony counts obtained from rectal samples of genetically lean and obese pigs fed rations containing low or high fiber Colony counts (cells x 19/g [dry wt] ± SE) from pigs fed rations containinga: Time on Low fiber High fiber diet (wk) Lean Obese Lean Obese 83.7 ± 8.7 114.8 ± 23.3 79.1 ± 9.12323b 121.6 ± 15.43b 3 64.4 ± 9.7 68.1 ± 9.8 35.1 ±4.1' 45.5 + 4.21.2 8 95.7 ± 1.3 93.7 ± 21.8 71.2 ± 8.21.23 122.9 ± 11.3 12 to 17 88.5 ± 31. 84.8 ± 4.1 9.5 + 6.72.3 76.9 ± 14.91.23 a Each value represents the mean number ± standard error of colony counts on TVC medium from three pigs (four roll tubes per pig). Means in each row do not differ (P >.5). Means in each column not followed by identical numerical superscripts differ (P <.5). Lean and obese pigs fed a low-fiber diet were slaughtered at 12 to 13 weeks; lean and obese pigs fed a high-fiber diet were slaughtered at 14 and 17 weeks, respectively. b Low-fiber diets. counts from the proximal to the distal end was obvious. The percentage of the total population metabolizing starch was 35 and 27% higher (P >.5) in the ceca and rectums, respectively, of lean animals fed the low-fiber diet than in those fed the high-fiber diet. In obese animals, the percentage of total counts found in the rectum that metabolized starch was 51% greater (P >.5) in pigs fed the high-fiber diet than in those fed the low-fiber diet. The percentage of total bacteria that metabolized the disaccharide of starch, i.e., maltose, was roughly similar for both diets in each genotype, with the exception that the counts from the ceca of lean pigs fed the high-fiber diet were 27% higher than in those fed the low-fiber diet. Other single energy sources used but not shown in Table 4 were mannitol, glycerol, lactate, and Trypticase. Each of these substrates supported less than 12% of the colonies observed with TVC medium and therefore are not reported here. Colonies in media without a substrate did not form to an appreciable size and could only be distinguished with a magnifying device. DISCUSSION The data from this study (Table 1) suggest that the microflora in the porcine gastrointestinal tract is initially suppressed when exposed to a high-fiber diet and that at a later time the flora reestablishes itself, suggesting that some adaptation takes place. The data in Table 2 suggest that adaptation of the cellulolytic population occurs more rapidly in lean-genotype than in obesegenotype pigs. The number of cellulolytic bacteria in lean pigs fed the high-fiber diet increased 17-fold, whereas a nonsignificant increase (P >.5) was observed in lean animals fed the lowfiber diet. This significant change (P <.5) also was not observed in obese animals on either diet. Whether the increase in the cellulolytic population is indirectly responsible for the increase in the total counts is uncertain. The cellulolytic bacteria cultured in this study at no time represented more than 2% of the total viable count. However, as a result of cross-feeding from the hydrolytic products of cellulose, i.e., cellobiose, glucose, etc., it is possible that the cellulolytic bacteria need to be present only in a catalytic concentration to greatly influence the total microbial count when a high-fiber diet is fed. Counts observed in the ceca and colons of lean pigs receiving the high-fiber diet were higher than in those receiving the low-fiber diet when glucose and cellobiose were used as the single energy sources (Table, thus supporting the potential cross-feeding hypothesis. Directly measuring cellulase activity would be beneficial Downloaded from http://aem.asm.org/ on July 4, 218 by guest TABLE 2. Comparison of mean most-probable-number counts of cellulolytic bacteria obtained from fecal samples of genetically lean and obese pigs fed rations containing low or high fiber Cellulolytic bacteria (MPN x 17/g [dry wt] + SE) from pigs fed rations containinga: Time on Low fiber High fiber diet (wk) Lean Obese Lean Obese 24. + 8.5' 11. + 3.7' 8.2 + 3.51b 76. + 41.1.2b 3 26. ± 6.41 18. ± 11.1 41. ± 17.1.2 94. + 47.12 8 12. ± 6.21 9.7 ± 5.91 141. ± 23.2 97. + 42.1.2 a Each value represents the mean most probable number (MPN) ± standard error of cellulolytic bacteria from three pigs. Means without a common numerical superscript differ (P <.5). blow-fiber diet.

11 VAREL ET AL. APPL. ENVIRON. MICROBIOL. C ' Cs 4...-I be 4c. 4 (- I- ' ' c) E m.a 4C.. 4. c ~ r_.2 be sco.1-5 C) e E C u C) cd '4 '4 u : '4 4 - E u :4 ' - m m~ % - as oo u VD r o N '. No '~. f.' ONo ~. - o o+ % % ' - % - O% 1. Nt - eni r N + '. ~ eo - %N \ ON ON N % tl +l a. N - ON " e +l +l o o 'It V-- O Chos L~ a i =~-=~.' co - ;> U o t- " o VI a., % - ei en N a - C) % - c. N % en ro e - + a. a,. r.' r '4. 'a 4 o. m.o E < X. mz C.).< A. m.1-1. wc cono. cis.5o. C- C3 ) o2 o X~ ~ * u. ' v dq 2 <2 2 8~ 3 C: > h '4 A:. : in determining the significance of this crossfeeding effect. Gargallo and Zimmerman (1) also found that cellulose digestion in pigs increased with time (12 to 28 to 4 days) when 1 or 18% of a basal diet was Solka-Floc. However, the study of Kass et al. (1, in which pigs were fed alfalfa meal and slaughtered at either 48 or 96 kg, showed that the apparent digestibility of nutrients in each section of the gastrointestinal tract was similar and suggested that any adaptive response to allow increased utilization of the fiber was minimal. No clear-cut distinction is implied in this study as to whether a higher total bacterial count is expected from pigs fed a highgrain diet than from those fed a high-forage diet, as might be expected in the rumen (16). The reason for the lower acetate-propionate ratio in the colon and rectum of both lines of animals fed the high-fiber diet than in those fed the low-fiber diet is unknown. The lower ratio was due to a significantly higher concentration (P <.5) of propionate production. The literature indicates conflicting results on the effect of fiber on acetate-propionate ratios in the gastrointestinal tracts of pigs (2, 8, 9, 13, 19). In the rumen, a lower concentration of propionate is expected when a high-fiber diet is fed; thus, the acetate-propionate ratio is increased. However, similarities in volatile acids between the rumen and the lower bowel are not necessarily comparable, owing to the different environments. In a study of the gastrointestinal traits of the animals used in this study (J. C. Pekas et al., Abstr. Am. Soc. Anim. Sci., abstr. no. 329, 1981) and a previous study (17), it was determined that the lean pigs were characterized by a heavier stomach and a significant increase in weight (P <.1) of the small intestine and of the colon-rectum when fed a high-fiber diet. The nature of this increased weight was believed to be hypertrophy of smooth-muscle linings of the gastrointestinal tract, but the microscopic morphology and biological importance were undetermined. These differences in gastrointestinal traits, whether negative or positive as far as nutrient efficiency is concerned, support the differences we observed with the cellulolytic microflora, i.e., that the responses of lean- and obese-genotype pigs to a high-fiber diet are different. The data in Table 4 indicate that, when specific energy sources were used to delineate the distribution of different bacterial populations in the cecum, colon, and rectum, different trends could be detected between high- and low-fiber diets. A consistent trend, i.e., differences in percentages of total counts cultured on specific energy sources in the cecum, colon, and rectum, was not always observed, perhaps implying in- Downloaded from http://aem.asm.org/ on July 4, 218 by guest

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112 VAREL ET AL. consistent methodology. However, Allison et al. (1) indicated that the variability they saw with counts from various pigs was due to actual differences in numbers of bacteria and not to the variability of the methodology for culturing bacterial cells. The data in Table 4 indicate that the percentage of total viable bacteria that metabolize a specific energy source declines from the proximal to the distal end of the large bowel, supporting the concept of Allison et al. (1) that bacterial populations from different sites in large bowels of pigs differ and that the extent of these differences (not yet fully known) may limit the conclusions that can be drawn from studies with fecal populations alone. We feel that the answer to both questions we were initially asking (i.e., does diet influence bacterial populations in the gastrointestinal tract, and do pigs of a widely different genetic background have different abilities to utilize dietary fiber?) is yes. However, further studies need to be conducted to determine the overall significance of these parameters. Additional microbiological studies need to be conducted to verify our results, along with nutritional studies to determine the performance of the host animal when exposed to a high-fiber diet. In the future, cannulation of the cecum and colon may prove beneficial in that numerous samples could be obtained instead of only one when the animal is slaughtered. Numerous studies (1, 15, 2-22) indicate animal-to-animal variability in bacterial populations; therefore, using a small number of cannulated animals initially fed one diet, sampled repeatedly, and then switched to another diet and sampled again may provide interesting results. ACKNOWLEDGMENTS We thank Ron Lindvall and his staff for animal care and feeding; Nancy Cook and her staff for animal slaughter; Lei Yen, Robert Lee, Sandy Fryda, and Moira Wilhelm for technical assistance, and Kathy Franje for stenographic work. The critical review and suggestions of Milton Allison and Tom Glass in the preparation of this manuscript are appreciated. LITERATURE CITED 1. Allison, M. J., I. M. Robinson, J. A. Bucklin, and G. D. Booth. 1979. Comparison of bacterial populations of the pig cecum and colon based upon enumeration with specific energy sources. Appl. Environ. Microbiol. 37:1142-1151. 2. Argenzio, R. A., and M. Southworth. 1975. Sites of organic acid production and absorption in the gastrointestinal tract of the pig. Am. J. Physiol. 228:454-462. 3. Betain, H. G., B. A. Linehan, M. P. Bryant, and L. V. Holdeman. 1977. Isolation of a cellulolytic Bacteroides sp. from human feces. Appl. Environ. Microbiol. 33:19-11. 4. Bornside, G. H. 1978. Stability of human fecal flora. Am. J. Clin. Nutr. Suppl. 31:5141-5144. 5. Bryant, M. P. 1972. Commentary on the Hungate technique for culture of anaerobic bacteria. Am. J. Clin. Nutr. 25:1324-1328. APPL. ENVIRON. MICROBIOL. 6. Bryant, M. P., N. Small, C. Bouma, and I. 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Types and distribution of anaerobic bacteria in the large intestine of pigs. AppI. Environ. Microbiol. 37:187-193. 22. Salanitro, J. P., I. G. Blake, and P. A. Muirhead. 1977. Isolation and identification of fecal bacteria from adult swine. AppI. Environ. Microbiol. 33:79-84. 23. Salyers, A. A., J. K. Palmer, and T. D. Wilkins. 1978. Degradation of polysaccharides by intestinal bacterial enzymes. Am. J. Clin. Nutr. Suppl. 31:5128-513. 24. Slavin, J. L., P. M. Brauer, and J. A. Marlett. 1981. Neutral detergent fiber, hemicellulose and cellulose digestibility in human subjects. J. Nutr. 111:287-297. 25. Steel, R. G. D., and J. H. Torrie. 196. Principles and procedures of statistics, p. 17-19. McGraw-Hill Book Co., New York. 26. Varel, V. H., A. G. Hashimoto, and Y. R. Chen. 198. Effect of temperature and retention time on methane production from beef cattle waste. Appl. Environ. Microbiol. 4:217-222. Downloaded from http://aem.asm.org/ on July 4, 218 by guest