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J. Anim Sci. 2008. 86:2979-2989. doi:10.2527/jas.2007-0819
© 2008 American Society of Animal Science
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ANIMAL NUTRITION

Comparative in vitro fermentation activity in the canine distal gastrointestinal tract and fermentation kinetics of fiber sources

G. Bosch1, W. F. Pellikaan, P. G. P. Rutten, A. F. B. van der Poel, M. W. A. Verstegen and W. H. Hendriks

Animal Nutrition Group, Wageningen University and Research Centre, PO Box 338, 6700 AH Wageningen, the Netherlands


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The current study aimed to evaluate the variation in fermentation activity along the distal canine gastrointestinal tract (GIT, Exp. 1). It also aimed to assess fermentation kinetics and end product profiles of 16 dietary fibers for dog foods using canine fecal inoculum (Exp. 2). For Exp. 1, digesta were collected from the distal ileum, proximal colon, transverse colon, and rectum of 3 adult dogs. Digesta per part of the GIT were pooled for 3 dogs, diluted (1:25, wt/vol), mixed, and filtered for the preparation of inoculum. A fructan, ground soy hulls, and native potato starch were used as substrates and incubated for cumulative gas production measurement as an indicator of the kinetics of fermentation. In addition, fermentation bottles with similar contents were incubated but were allowed to release their gas throughout incubation. Fermentation fluid was sampled at 4, 8, 12, 24, 48, and 72 h after initiation of incubation, and short-chain fatty acids and ammonia were measured. Results showed comparable maximal fermentation rates for rectal and proximal colonic inocula (P > 0.05). Production of short-chain fatty acids was least for the ileal and greatest for the rectal inoculum (P < 0.05). Therefore, for in vitro studies, fecal microbiota can be used as an inoculum source but may slightly overestimate in vivo fermentation. Experiment 2 evaluated the gas production, fermentation kinetics, and end product profiles at 8 and 72 h of incubation for citrus pectin, 3 fructans, gum arabic, 3 guar gums, pea fiber, peanut hulls, soy fiber, sugar beet fiber, sugar beet pectin, sugar beet pulp, wheat fiber, and wheat middlings. Feces of 4 adult dogs were used as an inoculum source. Similar techniques were used as in Exp. 1 except for the dilution factor used (1:10, wt/vol). Among substrates, large variations in fermentation kinetics and end product profiles were noted. Sugar beet pectin, the fructans, and the gums were rapidly fermentable, indicated by a greater maximal rate of gas production (Rmax) compared with all other substrates (P < 0.05), whereas peanut hulls and wheat fiber were poorly fermentable, indicated by the least amount of gas produced (P < 0.05). Sugar beet fiber, sugar beet pulp, soy fiber, and wheat middlings were moderately fermentable with a low Rmax. Citrus pectin and pea fiber showed a similar low Rmax, but time at which this occurred was later compared with sugar beet fiber, sugar beet pulp, soy fiber, and wheat middlings (P < 0.05). Results of this study can be used to formulate canine diets that stimulate dietary fiber fermentation along the distal GIT that may optimize GIT health and stimulate the level of satiety in dogs.

Key Words: dog • fiber • in vitro fermentation kinetics • short-chain fatty acid


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Although dogs have a relatively simple large intestine, they have an active microbial community that is capable of fermenting a significant quantity of dietary fiber (Fahey et al., 2004Go). This fermentation results in the production of mainly short-chain fatty acids (SCFA) that may affect host health. For example, acetate and propionate may stimulate the secretion of the satiety hormone peptide YY (PYY) by endocrine L-cells in the distal gastrointestinal tract (GIT; Karaki et al., 2006Go), and butyrate may protect against ulcerative colitis (Hague et al., 1997Go).

The largest fermentation activity is found in the proximal colon and declines further down the GIT when the availability of substrates decreases (Topping and Clifton, 2001Go). The presence of fermentable substrates affects microbial growth, and as a result of changing substrate availability, the microbial population changes in terms of species and numbers along the distal GIT (Marteau et al., 2001Go; Suchodolski et al., 2005Go). In relation to large intestinal health, it is of interest to stimulate degradation of dietary fiber and production of SCFA along the entire large intestine. It is thought that the kinetics and extent of fermentation of fiber indicate where the product is likely to be fermented in the GIT (Williams et al., 2005Go).

Although several in vitro studies have been conducted to evaluate the extent of fermentation of dietary fibers, little information is available regarding the fermentation kinetics of fibers for dogs. Moreover, the different in vitro studies used either ileal or fecal inoculum to characterize fermentation. The variation in the microbial community along the GIT questions whether inocula sources commonly used in literature are representative for the activity along the entire colonic region. The current study aimed to evaluate 1) the microbial fermentation activity in the canine lower GIT and 2) the fermentation kinetics of fibers for canine foods.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
The first study reported here was approved by and conformed to the requirements of the Ethical Review Committee for Animal Experiments of NOTOX BV (’s Hertogenbosch, the Netherlands).

Exp. 1

Substrates Three different types of dietary fiber (i.e., nondigestible oligosaccharides, cell-wall constituents, and resistant starch) were selected as potentially fermentable fiber sources to study the activity of fractions of the microbial population present in various parts of the GIT (Williams et al., 2000Go). Substrates were fructan (nondigestible oligosaccharides, Raftifeed IPS, Orafti, Tienen, Belgium), soy hulls, and native potato starch (both obtained from Research Diet Services, Wijk bij Duurstede, the Netherlands). Soy hulls were ground using a 1-mm sieve. Fructan and potato starch samples were already provided in a powdered form. Each substrate was analyzed for DM, ash, CP, and crude fat. Soy hulls were also analyzed for NDF, ADF, and ADL. The analyzed composition of each substrate is presented in Table 1Go.


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  		Table 1. Analyzed composition of the fibrous substrates used in Exp. 1 and 2
 
Digesta Collection This was an opportunistic study; inocula were obtained from dogs that were used in earlier experiments and scheduled for euthanasia. Three 2-yr-old healthy Beagle dogs (Marshall Bioresources, North Rose, NY) with a mean BW ± SEM of 9.6 ± 1.08 kg were housed individually and had free access to drinking water. Dogs were individually fed twice daily a commercially available dry extruded diet (Caro-croc Super Premium Lamb & Rice Diet, Vobra Special Petfoods BV, Veghel, the Netherlands) for 1 wk before digesta sampling. Dogs were fed approximately 500 kJ of energy/kg of BW0.75 per day. The diet was composed of dried lamb meat, rice, corn, dried beet pulp, beef fat, yeast, lamb meat extracts, vegetable oils, lecithin, vitamins, and micronutrients containing (g/kg of DM): 212 CP, 72 crude fat, 64 ash, 71 NDF, 39 ADF, and 14 ADL. Dogs were fed approximately 4 h before digesta sampling to ensure that sufficient digesta were available in the various intestinal segments for the preparation of the inoculum.

Dogs were anesthetized with an intravenous injection of 0.003 mg/kg of BW Fentanyl-Janssen (Janssen-Cilag BV, Tilburg, the Netherlands) and 50 mg/kg of BW Pentothal (Abbott BV, Hoofddorp, the Nether-lands). Subsequently, fatal incisions were made for exsanguination via the brachial and femoral arteries and veins in the groins of the front and hind legs. Immediately after euthanasia, the abdominal cavity was opened and 4 specific sections of the GIT of interest were clamped off to avoid mixing of digesta. Sections of interest were the terminal ileum (~15 cm proximal from the ileocecal junction), the proximal colon (~10 cm distal the ileocecal junction) including the cecum, the transverse colon (between the right and left colic flexures), and the rectum (~10 cm proximal from the anus). The GIT from stomach to anus was lifted out from the abdominal cavity, and each section with digesta contents was separated from the GIT. All materials used for digesta collection and storage were presterilized using 70% ethanol. Each section was placed on a glass plate and opened using a scalpel. The content of each section was transferred into sterile containers prefilled with carbon dioxide using a sterile spoon. All digesta samples were stored on ice (0°C) and transported to the laboratory of the Animal Nutrition Group (Wageningen University, Wageningen, the Netherlands).

Fermentation Procedures Methods used conformed to the procedures described by Williams et al. (2005)Go. Because in this study the variation among dogs was not of interest, digesta of the 3 dogs were quantitatively pooled to provide 1 representative inoculum for each GIT section for the specific diet (McBurney and Thompson, 1989Go). Digesta were diluted 1:25 by wet weight in a 39°C anaerobic sterile physiological saline solution (9 g/L of NaCl). The large dilution was necessary due to the limited quantity of material obtained. The diluted mixture was homogenized for 60 s using a hand blender and filtered through a double layer of sterile cheesecloth (16 threads/cm in both directions). The resulting filtrate was used as the inoculum. All procedures were carried out under a constant stream of carbon dioxide to maintain strictly anaerobic conditions.

Two batches of fermentation bottles were prepared. The first batch was used to measure the in vitro cumulative gas production for 72 h using an automated pressure evaluation system (APES; Davies et al., 2000Go). This technique measures the production of gas in the head space of bottles using pressure-sensitive switches and solenoid valves, which release a fixed amount of gas at a predetermined pressure threshold. The cumulative amount of gas produced during incubation time can be used to describe the kinetics of fermentation. To measure the production of fermentation end products over time, a second batch of fermentation bottles was incubated from which bottles were removed at regular time intervals (4, 8, 12, 24, and 48 h of incubation).

For the APES, a 5-mL sample of the pooled inoculum was injected into each 100-mL fermentation bottle (4 replicates per inoculum-substrate combination) and immediately attached to the APES. Each bottle contained approximately 0.5 g of substrate and 84 mL of a medium as described by Williams et al. (2005)Go. Incubation temperature was set at 39°C. Two blanks per inoculum containing only medium and inoculum were placed in the APES.

For measurement of fermentation end products, a 2.5-mL sample of the pooled inoculum was injected into a 50-mL serum bottle fermentation vessel. For each time point, at 4, 8, 12, 24, and 48 h of incubation, 4 replicates per inoculum per substrate were used. Each vessel contained approximately 0.25 g of substrate, 2.5 mL of inoculum, and 42 mL of a medium. For each time point, 2 blanks per inoculum containing only medium and inoculum were prepared. Each fermentation bottle was equipped with a rubber stopper through which a needle (BD Microlance, 0.45 x 13 mm, BD, Drogheda, Ireland) was pierced to release gas and placed in the incubator at 39°C. After inoculation of all fermentation bottles, each inoculum was analyzed for DM and ash.

Cumulative Gas Production Kinetics The monophasic model described by Groot et al. (1996)Go was fitted to the data for cumulative gas production [OM cumulative volume (OMCV) in mL of gas produced/g of OM weighed into the bottle] as follows: OMCV = A/[1 + (C/t)B], where A = asymptotic gas production; B = switching characteristic of the curve; C = time at which 50% of the asymptote had been reached; and t = time (h). The maximum rate of gas production (Rmax) and the time at which it occurred (Tmax) were calculated according to the following equations (Bauer et al., 2004Go): Rmax = {A x [CB] x B x [Tmax (–B–1)]}/{1 + [CB] x [Tmax(–B)]}2 and Tmax = C x {[(B – 1)/(B + 1)][1/B]}, respectively.

Chemical Analyses Dry matter and ash were determined by drying to a constant weight at 103°C and combusting at 550°C, respectively. Crude protein (6.25 x N) was determined using the Kjeldahl method (ISO, 2005Go), and crude fat was analyzed according to the Berntrop method (ISO, 1999Go). Neutral detergent fiber was analyzed according to a modified method of Van Soest et al. (1991)Go with a heat-stable amylase and expressed exclusive of residual ash, as described by Goelema et al. (1998)Go, and ADF and ADL were determined according to Van Soest (1973)Go.

Samples of fermentation liquids were analyzed for SCFA and ammonia. Short-chain fatty acids were determined using a gas chromatograph (Fisons HRGC Mega 2, Milan, Italy) equipped with a capillary column (Mega bore EC. 1000, internal diameter 0.53 mm, film thickness 1.0 µm, length 30 m, Alltech, Deerfield, IL). The ratio of the split injection used was 1:10. A flame ionization detector was used to identify the components within the sample. Column temperature was 110°C and was increased at a rate of 18°C/min up to 200°C in which T1 = 1 min and T2 = 2 min. Helium was used as a carrier gas at a flow rate of 8 mL/min with a 10-min run time for each sample. Isocaproic acid was used as an internal standard. Ammonia concentration of each fermentation liquid was determined by deproteinization of the supernatant using 10 g/L of trichloroacetic acid. Ammonia and phenol were oxidized by sodium hypochlorite in the presence of sodium nitroprusside to form a blue complex. The intensity of the blue color was measured colorimetrically at a wavelength of 623 nm

Statistical Analyses Overall effects of inoculum and substrate on the fermentation kinetics were tested for significance using ANOVA using the GLM procedure (SAS Inst. Inc., Cary, NC). The statistical model used was Y = µ + Ii + Sj + (I x S)ij + {varepsilon}ijk, where Y = variable to be tested; µ= mean; Ii = effect of inoculum i; Sj = effect of incubation substrate j; and {varepsilon}ijk = error term. For the fermentation end products, the statistical model also included incubation time as main effect and possible interactions of incubation time with inoculum and incubation substrate.

Exp. 2

Substrates Selection of substrates was based on their (potential) use in canine foods. Substrates used were citrus pectin (Herbacel AQ Plus type F, Herbafood Ingredients GmbH, Werder, Germany); fructans varying in chain length expressed as degree of polymerization (DP; fructan 1, Zopas, DP = 4; fructan 2, Raftifeed IPS, 2 < DP < 60; fructan 3, Beneo HP, DP > 22, all from Orafti, Tienen, Belgium); gums varying in botanical origin and viscosity (gum arabic, TIC Pretested Gum Arabic FT Powder, 0 to 300 centipoises (cP) of 30% solution; guar gum 1, TIC Pretested Pre-Hydrated Guar Gum 8/24 Powder, 4,000 to 6,500 cP of 1% solution; guar gum 2, TIC Pretested Gum Guar SCM Powder, 2,900 to 3,200 cP of 1% solution; guar gum 3, TIC Pretested Nutriloid 010 Powder, 0 to 200 cP of 2% solution, all from TIC Gums Inc., Belcamp, MD); pea fiber (Exafine 250, Cosucra SA, Warcoing, Belgium); peanut hulls (supplied by a company wishing to remain anonymous); soy fiber (Fibrim 1020 IP Non-GM, The Solae Company, Le Grand-Saconnex, Switzerland); sugar beet fiber (Fiberx 595, Danisco Sugar AB, Malmö, Sweden); sugar beet pectin (Pectin Classic RU 301, Herbstreith & Fox KG, Neuenbürg, Germany); unmolassed sugar beet pulp and wheat middlings (both Research Diet Services); and wheat fiber (Xylo-Gold Moon wheat fiber, Meneba Feed Ingredients, Rotterdam, the Netherlands). Substrates not already in a powder form were ground over a 1-mm sieve. Each substrate was analyzed for DM, ash, CP, and crude fat, whereas all substrates, except for the fructans and gums, were analyzed for NDF, ADF, and ADL. The analyzed composition of substrates used is presented in Table 1Go.

Feces Collection Four mature Labrador Retrievers with a mean BW ± SEM of 36.3 ± 2.51 kg from a local dog breeder were used as fecal donors. Dogs were fed twice daily the identical dry diet at the same concentration as used in Exp. 1 for 1 wk. The diet provided small amounts of fermentable fiber and thus avoided selection of microbes that had adapted to the substrates being tested in vitro (Bauer et al., 2003Go). Immediately after defecation, feces from each dog were collected and placed in a sterile plastic jar prefilled with carbon dioxide. All fecal samples were transported in a thermal insulated container within 10 min to the laboratory of the Animal Nutrition Group (Wageningen University, Wageningen, the Netherlands).

Fermentation Procedures Feces of the 4 dogs were pooled by weight and diluted 1:10 by wet weight in a 39°C anaerobic sterile physiological saline solution (9 g/L of NaCl) and further processed as described in Exp. 1. Two batches of fermentation bottles were prepared. The first batch was used to measure the in vitro cumulative gas production for 72 h using the APES and end product profile according to the procedures of Exp. 1. Because the concentrations of end products at 72 h may be more appropriate to describe the end product profile for slowly fermentable fibers (Awati et al., 2006Go), a second batch of fermentation vessels was used to evaluate the SCFA production for rapidly fermentable substrates. A preliminary experiment revealed that the most rapidly fermentable fibers reached Rmax at around 8 h of incubation. It was therefore decided to stop incubation after 8 h for measurement of SCFA concentrations. A 5-mL sample of the pooled inoculum was injected into a 100-mL serum bottle fermentation vessel (3 replicates per substrate) containing approximately 0.5 g of substrate and 84 mL of a medium. Two blanks containing only medium and inoculum were prepared. Each fermentation bottle was equipped with a rubber stopper through which a needle (BD Microlance, 0.45 x 13 mm, BD) was pierced to release gas and placed in the incubator at 39°C for 8 h. After incubation, all liquids were sampled and analyzed for SCFA and ammonia. After inoculation of all fermentation bottles, inoculum was analyzed for DM and ash.

Cumulative Gas Production Kinetics The monophasic model was used to describe cumulative gas production data. Equations to calculate model parameters Tmax and Rmax were the same as in Exp. 1.

Chemical Analyses Substrates were analyzed for composition using procedures described previously. Fermentation liquids were analyzed for SCFA and ammonia according to the procedures described in Exp. 1.

Statistical Analyses Overall effect of substrate on the parameters for fermentation kinetics was tested for significance using a 1-way ANOVA by the GLM procedure (SAS Inst. Inc.). The statistical model was Y = µ + Si + {varepsilon}ij, where Y = parameter to be tested; µ= mean; Si = effect of substrate i; and {varepsilon}ij = error term. The same model was used to evaluate the formation of fermentation products on 8 and 72 h of incubation separately. For each parameter, differences among means of substrates were tested for significance by ANOVA using the Tukey multiple range test. Individual means were considered different when they exceeded the minimum significant difference calculated by Tukey multiple range test.


   RESULTS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Exp. 1

Cumulative Gas Production Kinetics Incubation with the fructan samples yielded more OMCV compared with incubation with soy hulls or potato starch samples (Table 2Go). The decreased amount of gas produced from potato starch resulted in only a few measuring points, insufficient for the statistical program to fit the curve and allow estimation of Rmax and Tmax. Soy hulls showed less Rmax and greater Tmax than incubation with the fructan (P < 0.001 and P < 0.001, respectively). There was no difference in OMCV among inocula sources (P = 0.298). However, Rmax and Tmax were different (P = 0.028 and P < 0.001, respectively) among inoculum sources. The rectal inoculum had greater Rmax than the transverse colonic inoculum (P = 0.02). Compared with the large intestinal inocula, the ileum inoculum showed greater Tmax (P < 0.001). Interactions between inoculum source and substrate were not significant for OMCV (P = 0.245), whereas interactions for Rmax and Tmax were significant (P < 0.001 and P = 0.002, respectively). Ileal inoculum showed the fastest fermentation (i.e., greater Rmax and earlier time of Tmax) when incubated with soy hulls, whereas incubation with fructans resulted in the slowest fermentation compared with the other inocula (data not shown).


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  		Table 2. Fermentation parameters according to the effects of inoculum source and substrate (Exp. 1)
 
End Product Profile There was an effect (P < 0.001) of inoculum source, substrate, incubation time, and their interactions on fermentation end products (Table 3Go). The ileal inoculum produced the least amount of total SCFA per unit of OM throughout the incubation (P < 0.001). For the large intestinal inocula, total SCFA production was significantly less for the proximal and transverse colon compared with the rectum (P < 0.001 and P < 0.001, respectively). Incubation with the fructan samples resulted in the greatest production, whereas incubation with potato starch resulted in the least production of total SCFA per unit of OM (P < 0.001). Furthermore, fermentation of soy hulls resulted in the greatest ammonia production compared with the fructans and potato starch. All interactions between inoculum source, substrate, and incubation time were significant for production of acetate, propionate, butyrate, total SCFA, and ammonia (P < 0.001).


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  		Table 3. Concentration of fermentation products according to the effects of inoculum source, substrate, and incubation time (Exp. 1)
 
Exp. 2

Cumulative Gas Production Kinetics. Cumulative gas production per unit of OM differed among substrates (Table 4Go). Peanut hulls and wheat fiber resulted in the least OMCV (36.0 and 34.6 mL/g of OM, respectively; P < 0.05). Except for wheat middlings (151.2 mL/g of OM), all substrates resulted in OMCV values between 204.8 and 249.2 mL/g of OM. The decreased gas production with wheat fiber and peanut hulls resulted in missing model parameters such that the statistical program was unable to fit the curve and allow estimation of the parameters for fermentation kinetics. Sugar beet pectin, the fructans, and the guar gums showed increased Rmax in combination with decreased Tmax values. Among the 4 tested gums, incubation with gum arabic resulted in the greatest Tmax (P < 0.05). Sugar beet fiber, sugar beet pulp, soy fiber, and wheat middlings were moderately fermentable, indicated by a decreased Rmax (smaller than 10 mL/h). Similarly, citrus pectin and pea fiber showed a decreased Rmax, but Tmax was greater compared with sugar beet fiber, sugar beet pulp, soy fiber, and wheat middlings (P < 0.05).


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  		Table 4. Fermentation parameters for each of the 16 substrates (Exp. 2)
 
End Product Profile. At 8 h of incubation, sugar beet pectin showed the greatest production of acetate and total SCFA (P < 0.05; Table 5Go) compared with the other substrates. The 3 sources of fructans produced greater amounts of propionate and butyrate compared with sugar beet pectin (P < 0.05). Fructans produced the second greatest amount of total SCFA and the least amount of ammonia compared with the other substrates (P < 0.05). Among the 4 tested gums, large differences in total SCFA production were observed. The greatest amount of total SCFA was produced by guar gum 1 and guar gum 3 (P < 0.05). Fermentation of sugar beet fiber resulted in a greater amount of acetate compared with sugar beet pulp and citrus pectin (P < 0.05). Finally, compared with wheat fiber, incubation with wheat middlings resulted in similar amounts of total SCFA but significantly greater amounts of butyrate (P < 0.05).


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  		Table 5. Concentration of fermentation products at 8 and 72 h of incubation for each of the 16 substrates (Exp. 2)
 
At 72 h of incubation, the least SCFA production was detected for peanut hulls and wheat fiber (P < 0.05). Apart from incubation of wheat middlings, fermentation of the remaining substrates yielded SCFA values of approximately 10.5 mmol/g of OM. The 3 fructans and guar gums resulted in the greatest propionate production (P < 0.05). Wheat fiber and peanut hulls showed the least amounts of acetate and propionate (P < 0.05). Butyrate values were least for peanut hulls (P < 0.05). Incubation with pea fiber and wheat middlings resulted in the greatest butyrate production. The greatest amount of ammonia production was detected for wheat middlings (P < 0.05), whereas the 3 fructans and gum arabic showed the least ammonia values compared with all other substrates except guar gum 2 (P < 0.05).


   DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The present study is the first to provide insight into the fermentation activity of the microbial population along the canine distal GIT. Previous in vitro studies have investigated the fermentation characteristics of a wide range of fiber sources using canine ileal digesta (Zentek, 1995Go; Bednar et al., 2001Go; Murray et al., 2001Go) or feces (Sunvold et al., 1994Go, 1995aGo,bGo,cGo; Bednar et al., 2001Go; Swanson et al., 2001Go; Vickers et al., 2001Go) as the inoculum source. Studies using feces as the inoculum source made a general assumption that the fermentation by fecal microbes was representative for fermentation in the lower GIT. However, this assumption has not been tested. Fermentation characteristics of a dietary fiber depend on the physical and chemical composition of the fiber, the species and numbers of microbial community present in the GIT, and gastrointestinal transit time (Wong et al., 2006Go). Transit of digesta through the large intestine was shown to be related to body size and varied between 9 h for Miniature Poodles and 39 h for Giant Schnauzers (Hernot et al., 2006Go). In addition, there are community as well as numerical differences in the microbial population in the canine GIT. Suchodolski et al. (2005)Go reported similarities among the microbial populations in various parts residing in the canine GIT by comparing profiles of denaturing gradient gel electrophoresis. The ileum and colon had a similarity of 38.8%, colon and rectum showed 57.9% similarity, and ileum and rectum had the smallest denaturing gradient gel electrophoresis similarity of 32.8%. Numerically, the digesta in the ileum contains approximately 100 times less bacteria than the cecum (Gorbach et al., 1967Go), whereas the digesta in the cecum contains 250 times less total anaerobes compared with feces (Marteau et al., 2001Go). The inoculum source used in in vitro studies reported in literature, however, was not obtained from the location in the canine GIT where most fermentation can be expected (i.e., the colon) and may have over- or underestimated fermentability of dietary fibers.

Microbial fermentation activity in the present study differed among inoculum sources. In pigs, Williams et al. (1998)Go showed greater total SCFA production after 72 h of incubation with fecal inoculum compared with cecal inoculum. However, compared with large intestinal microbes, incubation of potato starch with small intestinal microbes resulted in greater in vitro gas and SCFA production values in pigs (Williams et al., 1997Go). Bauer et al. (2004)Go evaluated the microbial activity of cecal, midcolonic, and rectal inocula in pigs and showed that the amount of total gas produced and Rmax were greatest using inocula from the rectum, intermediate for midcolon, and the least for the cecum. The latter authors indicated that fecal inoculum used for in vitro fermentation studies could actually overestimate the microbial activity of the cecum. The ranking of the substrates, however, remained the same, independent of the large intestinal site from which the inoculum was obtained. This is consistant with observations made here in which an absolute difference in microbial activity among inoculum sources occurred. Large intestinal inocula showed slightly different fermentation kinetics, but each large intestinal inoculum site showed a similar ranking of the substrates based on their SCFA production over time (i.e., SCFA yield was greatest for fructan, intermediate for soy hulls, and least for potato starch). The latter conclusion has important implications for in vitro fermentation studies to evaluate large intestinal fermentability of dietary fiber sources for use in dog foods. Because the ranking remained the same for the large intestinal inocula, the use of feces for inoculum preparation appears to be suitable for in vitro screening purposes. However, because the total SCFA values for the proximal and transverse colonic inoculum were less than the rectal inoculum, in vitro studies may overestimate the extent of fermentation in these 2 sites when freshly voided feces are used. The use of ileal digesta as an inoculum source is not recommended to simulate fermentation activity by the proximal colonic microbial population. The decreased fermentation activity by the ileal microbial population shown in the present study in combination with the rapid transit time of digesta in the ileum (Hernot et al., 2006Go) does not make it likely that fermentation of fiber in the ileum is considerable in healthy dogs, although fermentation may begin there.

Fermentation of fructans with fecal inoculum resulted in SCFA production similar to values reported in other in vitro studies (Sunvold et al., 1995bGo; Vickers et al., 2001Go). For soy hulls, however, total SCFA values in the current study (2.49 mmol/g of OM at 12 h and 3.08 mmol/g of OM at 24 h) were approximately 2 to 3 times greater than those observed by Sunvold et al. (1995a)Go and Vickers et al. (2001)Go (1.02 and 0.84 mmol/g of OM at 12 h and 1.40 and 1.56 mmol/g of OM at 24 h, respectively). Potato starch has also been used as a substrate source but only with canine ileal digesta. Murray et al. (2001)Go showed a total SCFA content of 1.64 mmol/g of OM after 5 h of incubation with ileal digesta, which was considerably greater than the 0.64 mmol/g of OM reported by Bednar et al. (2001)Go after 7.5 h of incubation and the 0.38 and 0.37 mmol/g of OM reported in the present study at 4 and 8 h of incubation, respectively. There are no in vitro fermentation studies available in literature in which canine ileal or colonic digesta was used as an inoculum source and incubated with fructan or soy hulls. Differences among studies in SCFA yield may be related to variation in chemical composition of substrates, differences in composition of microbial communities among donors as affected by genetics (Zoetendal et al., 2001Go) or diet composition (Sunvold et al., 1995aGo), and methodological variations such as medium (Mould et al., 2005Go) and dilution factor for inoculum preparation (Bauer et al., 2004Go).

The present study is the first to provide characteristics of fermentation kinetics of a wide variety of dietary fibers, some commonly used in commercial canine diets. The fermentation kinetics may provide information on the likely site in the GIT where the fiber source will be fermented (Williams et al., 2005Go). Rapidly fermentable dietary fiber may be fermented more proximally in the GIT (ileum and proximal colon), whereas slower fermentable fibers are likely to also reach the more distal part of the GIT (e.g., distal colon). If fibers are very slowly fermentable, they will be voided in the feces before fermentation is complete. The production of specific end products of microbial degradation of dietary fiber at 8 and 72 h may provide insight into its potential to affect the host animal. For example, butyrate, the preferred energy source of colonocytes, can play an important role in the metabolism and normal development of colonic epithelial cells. This has been implicated in protection against ulcerative colitis (Hague et al., 1997Go). In addition, it should be noted that protein and AA from both undigested protein and proteins originating from endogenous secretions and desquamated mucosal cells of the GIT may be metabolized by the microbial population. If fermentable carbohydrate sources become depleted due to microbial degradation, fermentation becomes proteolytic, which may lead to the production of ammonia, branched-chain SCFA, amines, volatile phenols, and indoles, some of which can be toxic (Williams et al., 2001Go). Thus, it is of interest to supply saccharolytic sources of energy (dietary fiber) and production of SCFA from this source along the entire large intestine. Furthermore, SCFA may play a role in host satiety and appetite through its stimulation of GIT satiety hormones. For example, infusion of SCFA in rats (Cherbut et al., 1998Go) and fatty acids in dogs (Pappas et al., 1986Go) increased peripheral PYY concentrations. It is suggested that the SCFA (mainly acetate and propionate) activate a receptor (GPR43; Brown et al., 2003Go; Le Poul et al., 2003Go) expressed by endocrine L-cells, present predominantly in the canine distal GIT (Onaga et al., 2002Go), which are consequently stimulated to release PYY (Karaki et al., 2006Go). Stimulation of the secretion of glucagon-like peptide-1 (GLP-1), another satiety hormone produced and secreted by the endocrine L-cells (Kieffer and Habener, 1999Go), was also shown to be increased by the inclusion of fermentable fibers in diets of dogs (Massimino et al., 1998Go). It should be noted, however, that other characteristics of dietary fibers such as the ability to bind water and ability to affect viscosity of digesta may also contribute to the prolongation of satiety.

Cumulative gas production from the OM, Rmax, and Tmax as well as the end product profile at 8 and 72 h of incubation differed among substrates. According to the results of this experiment, sugar beet pectin, the 3 fructans, the guar gums, and gum arabic can be characterized as rapidly fermentable (large Rmax). Soy fiber, pea fiber, sugar beet fiber, sugar beet pulp, and wheat middlings were slowly fermentable. Wheat fiber and peanut hulls were poorly fermentable. For fermentation of gum arabic, it should be noted that this substrate showed significantly greater Tmax compared with the other rapidly fermentable substrates. It appeared that the substrate was poorly fermentable during the first part of incubation. The poor fermentability of gum arabic was also shown in other studies as indicated by reduced SCFA production at 6, 12, and 24 h of incubation (Sunvold et al., 1995aGo,bGo). The large Tmax may be due to a low amount of inoculated fecal microbes capable of degrading the substrates. After selective growth of these microbes during incubation, gum arabic appeared to be rapidly degradable, indicated by the large Rmax (34.2 mL/h). The data here suggest that gum arabic may also be rapidly fermentable in vivo but after adaptation to the substrate.

There was no effect of DP of tested fructans on fermentation rate or end product profile. Vickers et al. (2001)Go also did not find differences in SCFA production among fructans varying in DP. However, Roberfroid et al. (1998)Go reported that fructans with a DP <10 have a faster in vitro degradation rate compared with fructans with a longer chain length. Concerning the end product profile, rats fed a diet with fructans with a decreased DP showed increased butyrate concentration, whereas rats fed a diet with fructans with an increased DP give a greater concentration of propionate (Nilsson and Nyman, 2005Go). Furthermore, rats fed fructan-containing diets showed increased GLP-1 content and proglucagon mRNA in the proximal colon compared with controls with the short-chain inulin-type fructans (low DP) more potent to induce this effect (Cani et al., 2004Go). More in vivo studies are needed to demonstrate that the rate of degradation is actually different among fructans varying in DP.

Combined information about the fermentation kinetics and the individual SCFA produced at 8 and 72 h of a dietary fiber may be indicative of where the fiber is likely to be fermented in the GIT and where the end products (i.e., SCFA) may become available. Because dietary inclusion concentration of rapidly fermentable fibers is limited and some fermentation of dietary fiber may begin already in the ileum, the rapidly fermentable fiber source may become depleted, resulting in decreased amounts of fermentable carbohydrates more distal in the colon. The fiber sources characterized as slowly fermentable in vitro are expected to also be slowly fermentable in vivo and will yield moderate amounts of SCFA along the distal GIT. Howard et al. (2000)Go suggested that a combination of fiber sources could potentially be used to stimulate fermentation along the complete distal GIT in dogs. From the present study, this can be achieved by combining sugar beet pectin or fructans, which are rapidly fermentable and yield large amounts of acetate and propionate, with pea fiber or sugar beet pulp, which are slower fermentable and yielded large amounts of butyrate. The latter combination of fiber sources may be optimal not only for GIT health but also to stimulate the secretion of PYY and GLP-1. The latter could prolong feelings of satiety after a meal in dogs.

The present study shows that feces can be used as an inoculum source for in vitro fermentation studies but may slightly overestimate the actual fermentation processes occurring more proximally in the large intestine. The use of ileal digesta as an inoculum source is not recommended. The second experiment revealed large variation in the amount of fermentable components, the fermentation kinetics, and in the end product profile among tested fiber sources. Sugar beet pectin, the 3 fructans, the guar gums, and gum arabic were rapidly fermentable, whereas soy fiber, pea fiber, sugar beet fiber, sugar beet pulp, and wheat middlings were slowly fermentable. Wheat fiber and peanut hulls were poorly fermentable. Sugar beet pectin and the fructans resulted in the great amounts of acetate and propionate, whereas pea fiber, wheat middlings, and citrus pectin yielded greater amounts of butyrate. These results can be used to formulate canine diets to modulate the amount and site at which fermentation end products are generated to optimize not only GIT health but potentially also the level of satiety in dogs.

1 Corresponding author: guido.bosch{at}wur.nl

Received for publication December 20, 2007. Accepted for publication July 3, 2008.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 


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