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Nutrient digestibilities, microbial populations, and protein catabolites as affected by fructan supplementation of dog diets

Division of Nutritional Sciences and Department of Animal Sciences, University of Illinois, Urbana 61801

	Abstract

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Fructans are fermentable carbohydrates and include short-chain fructooligosaccharides (scFOS), inulin, and hydrolyzed inulin (oligofructose, OF). Two studies with dogs were designed to examine the effects of low concentrations of fructans on nutrient digestibilities, fecal microbial populations, and endproducts of protein fermentation, and fecal characteristics. In Exp. 1, 11 adult male beagles were fed corn-based, kibbled diets supplemented with or without OF to provide 1.9 ± 0.6 g/d. Dietary inclusion of OF decreased (P < 0.05) nutrient digestibilities, but did not affect fecal characteristics. Increasing OF concentration tended (P < 0.06) to linearly decrease fecal ammonia concentrations, but not those of branched-chain fatty acids (BCFA), amines, indole, or phenols. Fecal concentrations of total short-chain fatty acids (SCFA) and butyrate tended to be higher in OF-supplemented dogs (P < 0.10), as was the ratio of bifidobacteria to total anaerobes (P = 0.15). In Exp. 2, ileally cannulated adult female hounds were fed a meat-based kibbled diet and were assigned to four scFOS treatments (0, 1, 2, or 3 g/d) in a 4 x 4 Latin square design. Ileal nutrient digestibilities tended to increase (P < 0.15) with increasing concentrations of scFOS. On a DMI basis, fecal output tended to decrease linearly (P < 0.10) in response to increasing scFOS supplementation, whereas fecal score tended to exhibit a quadratic response (P = 0.12). In general, fecal concentrations of SCFA, BCFA, ammonia, phenols, and indoles were not altered by supplemental scFOS. Supplementation of scFOS increased fecal concentrations of total aerobes (P < 0.05) and decreased concentrations of Clostridium perfringens (P < 0.05). From these data, it seems that low levels of supplemental fructans have divergent effects on nutrient digestibility and fermentative endproducts, but do not adversely affect nutrient digestibility or fecal characteristics and may improve colonic microbial ecology in dogs.

Key Words: Digestion • Dogs • Feces • Fructans • Intestinal Microorganisms • Odors

	Introduction

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Excreta odor components are a subset of endproducts of protein fermentation and can be divided into five categories: ammonia in feces and urine; phenols and indoles in feces and urine; and branched-chain fatty acids (BCFA), amines, and volatile sulfur-containing compounds in feces (Miner and Hazen, 1969; Barth and Polkowski, 1974; Williams, 1984). These putrefactive compounds are the products of bacterial fermentation in the lower gastrointestinal tract (O’Neill and Phillips, 1992) and may be affected by dietary protein concentrations and/or individual AA themselves (Bakke, 1969a,b; Hobbs et al., 1996). Certain dietary carbohydrates may result in odor-reducing effects (Hidaka et al., 1986; Vince et al., 1990; Terada et al., 1992). Fructans are a class of fermentable carbohydrates that are indigestible by small intestinal enzymes (Hidaka et al., 1986; Roberfroid et al., 1993). Fructans include short-chain fructooligosaccharides (scFOS), inulin, and hydrolyzed inulin (oligofructose, OF). Whereas scFOS have a shorter chain length (degree of polymerization [DP] = 3 to 5), OF is composed of longer chains of fructose (DP of 3 to 10). In general, fructans are thought to inhibit either the production of protein-fermentative endproducts (excreta odor components) or the bacterial populations that produce them (Gibson and Roberfroid, 1995; Swanson et al., 2002).

The purpose of these studies was to elucidate the effects of selected dietary concentrations of scFOS and OF on nutrient digestibilities, gastrointestinal tract microbial populations, and fecal and urinary protein fermentation endproduct components of dogs.

	Materials and Methods

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Experiment 1

Experimental Design. Sixteen adult male beagles (mean age = 3 yr and mean BW = 12.0 ± 1.3 kg) were randomly assigned to one of four diets in order to create treatment groups with similar means and standard deviations in BW. Dogs were individually housed in stainless steel metabolism cages with wire mesh floors and drip pans for excreta collection. Dogs were kept in an environmentally controlled room with free access to water. All animal care procedures were approved by the Campus Laboratory Animal Care Advisory Committee, University of Illinois at Urbana-Champaign.

Dietary treatments consisted of four kibbled diets that were prepared under commercial conditions, with 0.9% OF (Raftifeed P75, Encore Technologies, Minnetonka, MN) added at the expense of corn prior to extrusion. The control diet had no added OF. Two intermediate diets (0.3 and 0.6% OF on a DM basis) were created by mixing the control and 0.9% OF diets (66.6% control and 33.3% OF diets to achieve 0.3% OF and 33.3% control and 66.6% OF to achieve the 0.6% OF diet). Individual meals were mixed in this manner to avoid kibble breakage that might occur in an industrial mixer. No variation was observed among the kibbles of the different diets. The ingredient composition of the basal diet is presented in Table 1. Diets were offered at 0800 and 2000 daily in 200-g portions with 100 g of water added to enhance acceptability.

Sample Collection. Individual samples of freshly voided feces were collected within 15 min of defecation and processed immediately in order to minimize any loss of volatile components. Fecal samples were weighed and mixed before measuring pH using an Accumet 1001 pH meter (Fisher Scientific, Inc., Pittsburgh, PA) with a MI-410 micro-combination pH electrode probe (Microelectrodes, Inc., Londonderry, NH). Feces were scored using a scale of 1 to 5, with 1 being dry, formed feces and 5 being a watery liquid. One aliquot of feces (5.0 g) was acidified with 25% m-phosphoric acid for short-chain fatty acids (SCFA), BCFA, and lactic acid analyses. Another fecal aliquot (2.0 g) was mixed with 6.0 mL of 6 N HCl for ammonia determination and stored at 4°C until analyses could be performed. For bacterial enumeration, a 1.0-g sample of feces was placed in a preweighed Carey-Blair transport media container (Meridian Diagnostics, Inc., Cincinnati, OH) and stored in liquid nitrogen until plating and enumeration could be performed. Fecal aliquots also were sealed in sterile sampling bags (Fisher Scientific) with excess air removed and stored at -20°C for subsequent analysis of amines, indoles, and phenols.

All remaining feces were weighed, scored, and collected twice daily for 4 d for determination of nutrient digestibilities. Samples were stored at -20°C until further processing. Urine samples were collected from each dog on three separate days by attaching a container with 10 mL of 6 N HCl to the drip pan in order to prevent ammonia volatilization and bacterial degradation. Urine samples were stored in glass bottles at 4°C for subsequent analyses.

Chemical Analyses. Feces were dried at 55°C prior to grinding in a Wiley mill through a 2-mm screen. Both DM and OM contents were determined according to AOAC (1984). Kjeldahl nitrogen (AOAC, 1984) and acid-hydrolyzed fat (Budde, 1952; AACC, 1983) also were determined, and their apparent digestibilities were calculated. Crude protein values were found by multiplying Kjeldahl N values by 6.25.

Fresh fecal samples (5.0 g) were acidified and diluted with 5.0 mL of 25% m-phosphoric acid and 15.0 mL of distilled water. After 30 min, samples were centrifuged at 25,000 x g for 20 min. The supernatant fluid was aspirated into microfuge tubes and frozen at -20°C. Following freezing, the supernatant was thawed, centrifuged at 13,000 x g for 10 min, and analyzed for lactic acid concentration (Barker and Summerson, 1941). Amounts of acetate, propionate, butyrate, valerate, isovalerate, and isobutyrate were determined using a Hewlett-Packard 5890A Series II gas chromatograph and glass column (180 cm x 4 mm i.d.) packed with 10% SP-1200/1% H₃PO₄ on 80/100 mesh Chromsorb W AW (Supelco Inc., Bellefonte, PA). Nitrogen was used as the carrier gas with a flow rate of 75 mL/min. Oven, detector, and injector temperatures were 125, 175, and 180°C, respectively. Concentrations of ammonia in feces and urine were determined using the method of Chaney and Marbach (1962). The specific gravity of urine also was measured using a mercury hydrometer.

For amine analysis, a modification of the Tabor and Tabor (1983) method was used. Feces (0.1 to 0.2 g, DM basis) were mixed with 5 mL of 10% (wt/vol) trichloroacetic acid and incubated at 4°C for 2 h with frequent mixing. Samples then were centrifuged at 29,000 x g for 20 min at 4°C. The supernatant was transferred and extracted three times with 5 mL of diethyl ether. The ether fraction was discarded, and residual ether was removed in vacuo. Amines were analyzed on a Beckman 6300 AA analyzer with postcolumn ninhydrin derivitization. The Tabor and Tabor (1983) rapid, single-buffer elution method was used to quantify putrescine, cadaverine, and spermidine. Histamine was not found to be a naturally occurring component of the feces and was chosen as an internal standard to determine the recovery of amines from the extraction procedure.

Indoles and phenols were extracted by mixing 2 g of feces with 5 mL of methanol containing 2,000 ppm of 5-chloroindole (internal standard). The feces-methanol mixture was covered with parafilm, mixed well, and incubated for 1 h at 4°C, with frequent mixing. Tubes then were centrifuged at 29,000 x g for 20 min at 4°C and the supernatant was collected. The remaining pellet was mixed again with 5 mL methanol and extracted as detailed above. The two supernatant fractions were combined for GLC analysis. Individual concentrations of indole, phenol, p-cresol, and 4-ethylphenol were determined using a Hewlett-Packard 5890A series II gas chromatograph and a Nukol fused-silica capillary column (60 m x 0.32 mm i.d.). Helium was used as the carrier gas with a flow rate of 100 mL/min. Oven temperature was 200°C and detector and injector temperatures were both 220°C.

Bacterial Enumeration. Total anaerobes, total aerobes, and bifidobacteria were determined by serial dilution of fecal samples in anaerobic diluent (Bryant and Burkey, 1953) before inoculation onto respective petri dishes of sterile agar. Total anaerobe and total aerobe agars were prepared according to Bryant and Robinson (1961) and Mackie et al. (1978). The selective medium for bifidobacteria spp. was anaerobically prepared with BIM-25 agar (BBL Microbiology Systems, Cockeyville, MD) according to the method described by Muñoa and Pares (1988).

All agars were inoculated by using a repeating pipette to dispense 7 drops of 10 µL each of the appropriate dilutions. After the drops adsorbed to the agar, plates were inverted and incubated at 38°C either anaerobically (for total anaerobes and bifidobacteria) or aerobically (for total aerobes). Colony counts were made after 48 h incubation. A colony-forming unit was defined as a distinct colony measuring at least 1 mm in diameter.

Statistical Analyses. Data were analyzed by ANOVA as a completely randomized design according to the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). Due to voluntary feed intake differences among treatments, OF consumption did not increase sequentially with OF level included in the diet. Therefore, data were analyzed by creating two treatment groups. The four control-fed animals all remained in the control group, whereas the 12 OF-fed dogs were grouped together. After removing five dogs due to outlying values in feed and OF consumption, the OF-supplemented group consumed a diet equivalent to 0.6% dietary OF (DM basis). Furthermore, DMI was used as a covariate in all statistical analyses. Least squares means are reported in tables. Although P < 0.05 was judged to be statistically significant, trends between P < 0.06 and P < 0.15 are also discussed.

Experiment 2

Experimental Design. Four purpose-bred, adult female dogs (Butler Farms USA, Clyde, NY) with hound bloodlines and an average weight of 20 ± 4 kg and age of 3 ± 1 yr were surgically prepared with ileal cannulas according to Walker et al. (1994). Dogs were housed in clean floor pens (1.2 x 3.1 m) in an environmentally controlled room (21°C and a 12-h light:12-h dark cycle) at the animal facility of the Edward R. Madigan Laboratory on the University of Illinois campus. All dogs were allowed free access to water. The surgical and animal care procedures were approved by the Campus Laboratory Animal Care Advisory Committee, University of Illinois at Urbana-Champaign.

Three concentrations of scFOS (1, 2, and 3 g/d) were tested against a control (no supplemental scFOS). Short-chain FOS (NutraFlora, GTC Nutrition Co., Johnstown, CO) was administered orally by gelatin capsule in order to avoid the possibility of scFOS degradation during diet processing. In addition to the scFOS, all animals consumed a dry, kibbled dog diet (Table 2), which was high in protein and fat concentrations, prepared under commercial conditions, and formulated to meet or exceed NRC recommendations for adult dogs at maintenance (1984).

Sample collection. During the collection phase, ileal effluent and feces were collected for 4 d. Ileal effluent was collected three times per day, with an interval of 4 h between collections. Individual ileal collections were 1 h long. Sampling times on the remaining 3 d were rotated 1 h from the previous day’s collection time. For example, on d 1, sampling took place at 0800, 1200, and 1600; on d 2, samples were collected at 0900, 1300, and 1700. Ileal samples were obtained by attaching a Whirlpak bag (Pioneer Container Corp., Cedarburg, WI) to the cannula barrel and around the cannula hose clamp with a rubber band. Prior to attachment of the bag, the interior of the cannula was scraped clean with a spatula and any digesta discarded. During collection of ileal effluent, dogs were encouraged to move around freely. Total feces excreted during the collection phase of each period was removed from the floor of the pen, weighed, composited, and frozen at -4°C. On d 14 of each period, a freshly voided fecal sample was collected for bacterial enumeration, as well as SCFA and ammonia determinations.

Samples were frozen at -4°C in their individual bags. At the end of the experiment, all ileal effluent samples were composited for each dog for each period and then refrozen at -4°C.

Chemical Analyses. Before analyses, ileal effluent was freeze-dried in a Tri-Philizer MP microprocessor-controlled lyophilizer (FTS Systems, Inc., Stone Ridge, NY). Diets and feces were oven dried at 55°C. Diets, feces, ileal samples, and freshly voided feces were prepared for chemical analyses as outlined in Exp. 1. Fecal amine measurement was performed using a different method as described for Exp. 1, which allowed for the detection of additional biogenic amines: agmatine, tryptamine, and spermine, and one monoamine, ethylamine. Both biogenic and monoamines were deriviatized with dansyl chloride and quantified using HPLC. Wet feces (2 g) were mixed with 15 mL of 0.4 N perchloric acid and 0.04 mL of 5,000 ppm of 1,6-hexanediamine dihydrochloride (internal standard). Samples were centrifuged at 2,000 x g for 10 min. After aspirating the supernatant, the remaining fecal pellet was mixed with 7 mL of 0.4 N perchloric acid and centrifuged at 2,000 x g for 10 min. The supernatants were combined and centrifuged at 12,500 x g for 5 min. The supernatant was aspirated and mixed with 0.2 mL of 2 N NaOH, 0.3 mL of saturated Na₂CO₃, 2.0 mL of 10 mg/mL dansyl chloride, and incubated at 40°C for 45 min. Then, 0.2 mL ammonium hydroxide was added and tubes were incubated again at 40°C for 45 min. Acetonitrile (1.3 mL) was added and tubes were centrifuged for 5 min at 2,000 x g. For biogenic amines, 10 µL of each sample was injected into a Dionex BioLC HPLC (Dionex Corp., Sunnyvale, MA) equipped with a NovaPak C18 column (150 x 3.9 mm, 4 µm i.d.) and NovaPak C18 guard (Waters Corp., Milford, MA). The mobile phase (1.0 mL/min) consisted of acetonitrile and 100 mM ammonium acetate that was ramped from a 30:70 ratio to a 90:10 ratio of acetonitrile and ammonium acetate, respectively. The cleaning cycle consisted of 100% acetonitrile and the column was reequilibrated with 30:70 acetonitrile:ammonium acetate for a total run time of 56.9 min. Ultraviolet detection at 254 nm was employed to detect agmatine, cadaverine, putrescine, spermidine, spermine, and tryptamine. Monoamines (100 µL) were analyzed on a Dionex DX300 HPLC fitted with a Lichrosorb RP8 column (250 x 4 mm, 10 µm i.d.) and a Lichrosorb RP8 guard column (Phenomenex, Torrance, CA). The mobile phase (1.0 mL/min) consisted of 55% methanol in water and was ramped up to 90% methanol prior to a cleaning cycle of 100% methanol and reequilibration with 55% methanol for a total run time of 45 min. Ultraviolet detection at 254 nm was employed, and ethylamine was the only monoamine detected.

Diets, feces, and ileal samples were analyzed for DM, OM, ash, CP, and acid-hydrolyzed fat as described in Exp. 1. Feces and ileal samples also were analyzed for chromium content according to Williams et al. (1962) using an atomic absorption spectrophotometer (model 2380, Perkin-Elmer, Norwalk, CT). Short-chain fatty acids, BCFA, phenol, and indole concentrations, and fecal DM percentage and pH were measured in fresh fecal specimens as described for Exp. 1, but lactate concentrations were not measured.

Bacterial Enumeration. Total anaerobes, total aerobes, bifidobacteria, lactobacilli, and Clostridium perfringens were determined by serial dilution of fresh fecal samples in anaerobic diluent and inoculation onto respective petri dishes of sterile agar as described in Exp. 1. Lactobacilli were cultured on Rogosa SL agar (Difco Laboratories, Detroit, MI). Clostridium perfringens was enumerated on a tryptose-sulfite-cycloserine agar with egg yolk (FDA, 1992). Plates were incubated anaerobically prior to enumeration of cfu as outlined in Exp. 1.

Statistical Analyses. Data were analyzed by ANOVA of the GLM procedure of SAS. Model sums of squares were separated into treatment, period, and animal effects. Treatment means were compared using linear and quadratic contrasts (Steele and Torrie, 1980). Additionally, a contrast was constructed to compare all levels of scFOS supplementation vs. the control. As before, P < 0.05 was judged to be statistically significant, but trends between P < 0.06 and P < 0.15 also are discussed.

	Results and Discussion

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Experiment 1

Diet Composition and Effects of OF on Nutrient Intake and Digestibility. Both experimental diets had similar DM (96.2%), OM (91.1%), CP (24.8%), and lipid (7.1%) contents (data not shown). Nutrient intakes did not differ for DM (258 vs. 301 g/d), OM (237 vs. 297 g/d), CP (63 vs. 83 g/d), and lipid (20 vs. 23 g/d) intakes between treatment groups (data not shown). Although treatment mean intakes were not different, there was wide variation in DMI (pooled SEM = 74.6). Since OF was an integral part of the diet, DMI significantly affected response variables and was therefore used as a covariate in statistical analyses. Calculated OF consumption in OF-supplemented dogs was 1.9 ± 0.6 g/d (range = 1.1 to 2.6 g/d). Total-tract digestibilities of DM, OM, and lipid were lower in OF-supplemented dogs (P < 0.05, 0.05, and 0.01, respectively; Table 3). Total-tract digestibility of CP tended (P = 0.07) to be lower for OF-supplemented animals compared with unsupplemented animals. These results are similar to those found by Propst and coworkers (unpublished data), who reported that supplementing dog diets with OF at 0.3, 0.6, or 0.9% (DMB basis) decreased (P < 0.05) total-tract digestibility of DM, OM, and CP. However, in that study, lipid digestibility was unaltered by OF supplementation. The current results are in contrast to Diez et al. (1997), who reported that 4 or 8% dietary OF (DM basis) did not reduce total-tract digestibility of DM, OM, or ether extract for dogs fed a beef-, corn-, and vegetable oil-based diet. However, Diez and coworkers (1997) did report that 8% dietary OF reduced digestibility of CP (83.8 vs. 87.8% for supplemented and control diets, respectively). In the current study, it is unlikely that very much of the decrease in DM and OM digestibility can be attributed to incomplete fermentation of OF since fructans are readily fermentable by the canine microflora (Sunvold et al., 1995a) and composed a small portion of the total diet. It is possible, however, that the decrease in nutrient digestibility was due to a decrease in intestinal transit time, a common attribute of fermentable fibers (Fahey et al., 1990; Lewis et al., 1994). The tendency of OF to reduce CP digestibility is likely due to an increase in bacterial cell synthesis. Wolf et al. (1998) proposed that as the percentage of fermentable carbohydrate increases in the diet, the amount of microbial mass increases in the feces. This would result in an increase in fecal N excretion and a decrease in apparent CP digestibility. The decrease in lipid digestibility was surprising and suggests that OF may interfere with lipid digestion and absorption or that OF may have complexed with dietary fat during extrusion, decreasing digestibility.

Fecal Short-Chain Fatty Acid Concentrations. Fecal concentrations of acetate and lactate did not differ due to OF supplementation (Table 5). Propionate concentrations were increased (P < 0.05) in feces of OF-fed dogs. Fecal concentrations of butyrate and total SCFA tended to be greater (P = 0.15 and 0.07, respectively) in OF-fed dogs. Similar increases were reported by Propst and coworkers (unpublished data) in dogs fed a poultry byproduct- and rice-based diet supplemented with 0.3, 0.6, or 0.9% dietary OF. The increase in fecal concentrations of SCFA is probably due to an increase in the amount of readily fermentable substrate in the OF-supplemented diet. Also, due to their longer DP, OF are fermented more slowly than are scFOS (Rycroft et al., 2001; Perrin et al, 2002), perhaps leading to more SCFA production in the distal portion of the colon, and therefore, appearance in feces. This increase in SCFA, especially butyrate, is viewed as a positive result because of the role of butyrate and other SCFA in providing energy for colonic epithelial cells (Roediger 1980; Sakata 1987).

Nutrient Intakes and Ileal and Total-Tract Digestibilities. The basal diet fed to all dogs contained, on average, 92.7% DM, 93.2% OM, 33.6% CP, and 22.5% lipid. A single dog exhibited nutrient digestibility values that were greater than three standard deviations lower than the other animals. Therefore, this animal’s nutrient intake and digestibility data were excluded from statistical analysis. Mean intakes of DM, OM, CP, and lipid were similar among all treatment groups (Table 8). Ileal digestibilities of DM (P = 0.14), OM (P = 0.13), CP (P = 0.09), and lipid P = 0.07) tended to increase in a linear fashion in response to scFOS concentration. These results are in contrast to Swanson et al. (2002), who reported that 2 g/d scFOS did not significantly alter ileal digestibility of DM, OM, or CP in dogs fed a poultry byproduct meal- and rice-based diet. A possible explanation for this deviation in results is that Swanson’s control diet, although similar in composition, had higher ileal DM digestibility (67.7%) compared with our control diet (60.3%).

Fecal Characteristics and Ammonia Concentrations. When expressed on a g/d basis, neither wet nor dry fecal outputs were changed in response to scFOS supplementation (Table 9). However, on a g/g of DMI basis, both wet and dry fecal outputs tended to decrease linearly (P = 0.09 and 0.06, respectively) in response to increasing scFOS supplementation. Fecal DM percentages did not differ among treatment groups. This is supported by Diez et al. (1998), who determined that 5% dietary OF did not alter dry fecal output and decreased fecal DM percentage for dogs consuming a beef- and rice-based diet. Swanson et al. (2002) did not detect any significant changes in fecal DM in dogs supplemented with 0 or 2 g of scFOS/d either. Fecal score tended to exhibit a quadratic (P = 0.12) response to scFOS supplementation and was highest in dogs receiving 1 g/d scFOS and lowest in dogs receiving 3 g/d scFOS. A higher fecal score indicates formation of a softer stool. It should be noted, however, that all fecal scores remained within an acceptable range. Fecal pH also responded in a quadratic fashion due to scFOS supplementation and was highest in dogs receiving 1 g/d scFOS and lowest in dogs receiving either 0 or 3 g/d scFOS. A reduction in fecal pH generally indicates increased colonic fermentation. Fecal concentrations of ammonia did not differ due to scFOS supplementation. This is similar to the results reported by Swanson et al. (2002), which indicated that 2 g/d scFOS did not significantly alter fecal score, fecal pH, or fecal concentrations of ammonia.

Fecal Bacterial Concentrations. Total anaerobes, bifidobacteria, and lactobacilli concentrations in feces were not altered by supplemental scFOS (Table 12). Total aerobe concentrations were linearly increased (P < 0.05) by FOS consumption. The increase in total aerobes was unexpected, and may be partially attributed to the relatively low level of total aerobes in the control group. Fecal concentrations of Clostridium perfringens tended to decrease (P = 0.08) when comparing all scFOS-supplemented dogs to the control. In contrast to the previous study, the ratio of bifidobacteria to total anaerobes was not altered due to scFOS supplementation. Swanson et al. (2002) also found no change in fecal bifidobacteria or lactobacilli due to supplementation of 2 g/d scFOS to adult dogs.

Differences in results of these two studies may be due to a number of factors. One of these differences is in basal diet fed. The first study utilized a corn-based diet containing 24.8% CP and 7.1% fat, whereas the second study utilized a meat-based diet with considerably higher CP (33.6%) and fat (22.5%) content. Diets rich in protein have been implicated as a factor in increasing fecal odor components, many of which are endproducts of protein fermentation (Williams, 1959; Bakke, 1969a,b; Tabor and Tabor, 1985). In the present two studies, not all odor components responded in this predictable fashion. In the first study (with lower dietary CP), although fecal concentrations of amines and ammonia were lower than in the second study, BCFA, indoles, and phenols were higher than in the second study. This may be due in part to the digestibility of the protein sources found in these diets. Although direct statistical comparisons cannot be made, numerically, CP in the plant-based diet used in Exp. 1 likely was less digestible than the CP in the animal-based diet in Exp. 2. Accordingly, the type of AA reaching the large intestine and available for fermentation may alter production of fecal odor components. Tryptophan is the primary precursor for indole, tyrosine is the precursor of phenol, and arginine precedes putrescine; putrescine and methionine combine to form spermidine and spermine (Tabor and Tabor, 1984). Unfortunately, AA digestibility was not measured in either study.

In addition to differences in CP level and digestibility, the diets employed in these studies had very different levels of OM digestibility and, in turn, dietary fiber. The reduced OM digestibility of the diet used in Exp. 1 indicates more fermentative substrate was available to the colonic microflora and may have influenced its composition.

Another factor to consider is that the basal diet used in the first study contained approximately 15% wheat grain, which contains an average of 1.4 mg/g of scFOS (Campbell et al., 1997). Calculated, this would contribute 0.21 mg of scFOS/g of diet, or about 0.02% dietary scFOS in addition to the supplemented levels of OF.

An additional factor contributing to the differences found in these two studies is the different type of fructan supplemented. The first study supplemented diets with OF, which is a hydrolysis product of inulin and which has an average DP of 6 sugar units, although longer chains of fructans are also present. The second study supplemented diets with scFOS, which is produced enzymatically from sucrose and has an average DP of 4. Furthermore, the two studies used different methods of administering fructans. The first study incorporated OF into an extruded diet. The extrusion process may have caused some degradation or complexing of the OF. The second study administered scFOS orally via gelatin capsule in order to prevent any losses during diet manufacturing. Also, this method of administration allows for a constant level of fructan intake, despite differences among treatment groups in basal diet consumption.

Finally, one cannot ignore the fact that differences in individual animals (breed, gender) and in the experimental design employed between these two studies may have contributed to the divergent results. For example, dogs in Exp. 1 had much higher fecal concentrations of total aerobes than did dogs in Exp. 2. This may have contributed to the increase in total aerobes in the second study, whereas the first study elicited a decrease. Furthermore, although control group fecal bifidobacteria concentrations were similar between the two studies (9.4 vs. 9.5 log₁₀ CFU/g of feces DMB) and fructan supplementation resulted in similar increases in this bacteria type (9.5 vs. 9.8 log₁₀ CFU/g feces of DMB), only Exp. 1 detected this as a statistically significant result. This may be explained by differences in standard errors of the mean (0.15 vs. 0.30) and to the experimental methods utilized (11 animals in a completely randomized design for Exp. 1 and 4 animals in a Latin square design for Exp. 2).

	Implications

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The results of these two studies illustrate that different forms of fructans can have different physiological effects in dogs. Specific effects may vary due to fructan chain length and/or rate of fermentation. Another important suggestion from these studies is that the type of basal diet utilized (i.e., plant-based vs. animal-based; level of crude protein) and variation among individual animals might greatly affect the efficacy of fructan supplementation. These data suggest that future studies should carefully examine the individual effects of fructan form, basal diet composition, and animal attributes. This will allow the scientific community to elucidate the specific effects of fructans in dog nutrition, elicit more consistent results, and determine which factors are the most appropriate to consider for fructan inclusion in canine diets.

	Footnotes

 
¹ Present address: Lobeliastraat 110, 1616 XM Hoogkarspel, The Netherlands.

² Present address: School of Veterinary Medicine, University of Nevada-Reno, Mailstop 202, Reno 89557.

³ Correspondence: 166 Animal Sciences Laboratory, 1207 W. Gregory Dr., Urbana, IL 61801 (phone: 217-333-2361; fax: 217-244-3169; E-mail: g-fahey{at}uiuc.edu).

Received for publication February 22, 2002.

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