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Growth performance, gastrointestinal microbial activity, and nutrient digestibility in early-weaned pigs fed diets containing flaxseed and carbohydrase enzyme^1,2

E. Kiarie^*,3, C. M. Nyachoti^*, B. A. Slominski^* and G. Blank

^* Departments of Animal Science and and Food Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada

	Abstract

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The effects of ground flaxseed (FS) and a multicarbohydrase enzyme (C) supplement on piglet performance, gastrointestinal microbial activity, and nutrient digestibility were investigated in a 28-d trial. The enzyme supplement provided 500 units of pectinase, 50 units of cellulase, 400 units of mannanase, 1,200 units of xylanase, 450 units of glucanase, and 45 units of galactanase per kilogram of diet. Ninety-six pigs were weaned at 17 d of age (BW, 6.1 ± 0.4 kg, mean ± SD) and assigned to treatments based on a 2 x 2 factorial arrangement in a completely randomized design, with 6 pens per diet (4 pigs per pen). The diets contained wheat, barley, peas, soybean meal, and canola meal with 0 or 12% FS, and were fed without or with C. Flaxseed was included by changing the levels of the other ingredients to balance the diets for DE and nutrients. Diets had similar nutrient contents and met the NRC (1998) nutrient specifications, with the exception of DE, CP, and AA, which were 95, 94, and 97% of the NRC requirements, respectively. Diets were fed in a 2-phase feeding program (2 wk/phase). Feed intake and BW were measured weekly, and 1 pig per pen with a BW nearest the pen average was bled weekly to evaluate plasma urea nitrogen. On d 28, fresh fecal samples were collected from each pen and 1 pig per pen with a BW nearest the pen average was killed to evaluate intestinal microbial activity and nutrient digestibility. A dietary effect on piglet performance was observed only in wk 3, when the FS diets decreased (P = 0.005) ADG and G:F, tended to decrease (P = 0.070) ADFI, and increased (P = 0.027) plasma urea nitrogen. An interaction between FS and C was observed for ileal digesta viscosity (P = 0.045), such that C increased viscosity in the FS diet but had no effect in the non-FS diet. Flaxseed and C interacted to affect ileal ammonia content (P = 0.049), such that in the absence of FS, pigs fed the diet with C had lower ammonia than those on the diet without C. Flaxseed and C affected other ileal parameters independently. Pigs fed the FS diets had decreased (P = 0.003 to 0.033) anaerobic spore counts, organic acid, DM, CP, and nonstarch polysaccharide (NSP) digestibility compared with pigs fed the non-FS diets, whereas pigs fed the C-supplemented diets had greater (P = 0.009 to 0.061) lactobacilli counts, lactate, DM, and NSP digestibility than pigs fed the unsupplemented diets. In conclusion, FS reduced ileal microbial activity, nutrient digestibilities, and piglet performance in wk 3. The multicarbohydrase supplement increased ileal DM and NSP digestibilities as well as lactobacilli counts and lactate.

Key Words: carbohydrase • early-weaned pig • flaxseed • growth performance • intestinal microbial activity • nutrient digestibility

	INTRODUCTION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Weaning exposes piglets to nutritional and environmental stresses, resulting in reduced performance and, in some instances, diarrhea and death (Cromwell, 2002). Growth performance at this stage is optimized by the use of in-feed antibiotics (Pluske et al., 2002). Because of concerns with residues in meat products and bacterial resistance to antibiotics, investigating alternatives to in-feed antibiotics is required (Cromwell, 2002).

Dietary manipulation has been proposed as a strategy for optimizing the performance of newly weaned pigs (Pluske et al., 2002). Flaxseed (FS) is a rich source of -linolenic acid (ALA) and lignans, which possess broad antimicrobial activity (Pauletti et al., 2000; Kankaanpää et al., 2001). It can be speculated that including FS in weaned pig diets could potentially modulate intestinal microbiota (Smith et al., 2004). However, few studies have assessed the effects of FS on intestinal microbial activity in weaned pigs because most swine research on FS has primarily focused on changing the fatty acid profile in finishing pigs (e.g., Mathews et al., 2000).

A concern with using FS in piglet diets is the high content of mucilaginous nonstarch polysaccharide (NSP), which could increase digesta viscosity and thus impair nutrient utilization (Bhatty, 1993). However, supplemental carbohydrase enzymes, which have long been recognized as effective in hydrolyzing NSP in feed-stuffs for swine (Kim et al., 2003; Omogbenigun et al., 2004), may allow inclusion of FS in piglet diets. Furthermore, Vahjen et al. (1998) has suggested that carbohydrase enzymes may partially hydrolyze NSP in the intestinal tract to yield substrates capable of modulating microbial activity.

Therefore, we studied the effects of supplementing starter diets with flaxseed and a multicarbohydrase enzyme on growth performance, gastrointestinal microbial activity, and nutrient digestibility in early-weaned pigs.

	MATERIALS AND METHODS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Experimental Diets
The diets were based on wheat and barley as the cereal source and pea, soybean, and canola meals as the protein source, with 0 or 12% ground FS. The diets were fed with or without a multicarbohydrase enzyme supplement (C). The C preparation supplied, per kilogram of complete diet, 500 units of pectinase, 50 units of cellulose, 400 units of mannanase, 1,200 units of xylanase, 450 units of glucanase, and 45 units of galactanase. Diets were formulated for a 2-phase feeding program for early-weaned pigs (phase I, 1 to 14 d; and phase II, 15 to 28 d) corresponding to the NRC (1998) nutrient specifications for pigs weighing 5 to 10 kg and 10 to 20 kg of BW, respectively. The diets for phase I were fortified with dried whey, lactose, and oat groats to ease the transition from the sow’s milk. Diets were formulated to meet the NRC (1998) requirements, with the exception of DE, CP, and AA, which were 95, 94, and 97% of NRC requirements, respectively (Table 1). Prior to diet mixing, FS was ground in a hammer mill to pass through a 3-mm screen. Ground FS was added in the diet by changing the levels of wheat, barley, wheat middling, pea, soybean, and canola meals to balance the diets for DE and nutrients. Chromic oxide (0.3%) was added to each diet as an indigestible marker, and the diets were provided in mash form. Flaxseed used in the current study was provided by the Flax Council of Canada (Winnipeg, Manitoba, Canada).

Performance Monitoring and Sample Collection
Experimental diets were fed for a 28-d period, during which individual BW and feed disappearance were monitored weekly. On d 7, 14, 21, and 28, blood samples (10 mL) were collected, from 1 pig per pen whose BW was nearest the pen average, via jugular venipuncture into Vacutainer tubes coated with lithium heparin (Becton Dickinson & Co, Franklin Lakes, NJ). The samples were immediately centrifuged at 2,000 x g for 10 min at 5°C to recover plasma, which was immediately stored at –20°C until used for plasma urea N (PUN) analysis.

Freshly voided feces were collected from each pen on d 28 for determination of nutrient digestibility, pH, and ammonia. Fecal pH was determined prior to acidifying the samples with 0.1 N HCl (1:1, wt/vol). Samples were frozen at –20°C until required for analysis. After 28 d, pigs were put under feed restriction for 3 d prior to slaughter. Feeding time was tailored to the estimated slaughter time, allowing 45 min per pig for anesthetizing (see below) and collecting samples. The daily feed allowance was calculated to provide 2.6 times the maintenance energy, which was assumed to be 110 kcal of DE per kg of BW^0.75 (NRC, 1998) and based on the pen average BW on d 28. Feed intake was restricted to maximize recovery of the digesta at the terminal ileum (Houdijk et al., 2002).

At the end of the 3-d restricted-feeding period, 1 pig per pen with a BW close to the pen average was killed to evaluate digesta pH, selected bacterial populations, viscosity, organic acids, nutrient digestibility, and ammonia content. On the slaughtering day, pigs were fed at the designated time; 2 h later, pigs were held under general anesthesia by inhalation of 5% isoflurane (Pharmaceutical Partners of Canada Inc., Richmond Hill, Ontario, Canada) via a facial mask.

The abdominal cavity was exposed by midline incision, and the ileal-cecal junction was located; the ileum with its contents was immediately clamped at 30 and 5 cm cranial to the junction, freed of mesentery, cleaved, and flushed with physiological saline (Bimeda-MTC Animal Health Inc., Cambridge, Ontario, Canada) to remove excess blood. Ileal luminal contents were emptied into a sterile, plastic sample bag (Fisher Scientific, Fair Lawn, NJ) and gently mixed. Approximately 5 g of the sample was aseptically transferred into a second sterile bag and placed on ice and transported (within 1 h) to the laboratory for selected bacterial enumeration. The viscosity and pH of the remaining ileal samples were determined, after which the samples were acidified (0.1 N HCl, 1:1 wt/vol) and stored at –20°C until used for analysis. The entire cecum and midcolon (30 cm) were bisected and flushed with physiological saline, and the contents from each segment were emptied into separate sterile bags. The pH was determined, and the samples were acidified (0.1 N HCl, 1:1 wt/vol) prior to storage at –20°C until used for analysis. Following digesta sampling, pigs were killed with an intracardiac overdose (110 mg/kg) of sodium pentobarbital (Bimeda-MTC Animal Health Inc.).

Digesta and Fecal pH Measurements
The pH of the ileal, cecal, and colon digesta were measured on undiluted samples. Fecal pH was measured on fecal homogenate (feces:deionized water, 1:1 wt/vol). All pH measurements were made with an electronic pH meter (Accumet Basic, Fisher Scientific), which was standardized with certified pH 4 and 7 buffer solutions.

Ileal Digesta Viscosity Measurements
Ileal digesta samples from some pigs were too dry, and it was difficult to obtain more than 0.1 mL of supernatant sample for viscosity measurement. Therefore, all ileal samples were diluted 1:1 (wt/vol) with deionized water to enable comparison among dietary groups. Briefly, within 30 min of collection, 1 g of thoroughly mixed ileal contents was diluted 1:1 (wt/vol) with deionized water, vortex mixed, and centrifuged at 12,000 x g for 8 min. The supernatant fraction (0.5 mL) was placed in a Brookfield digital viscometer (Model DV-II+ Version 3.0, Brookfield Engineering Laboratories Inc., Stoughton, MA), in which viscosity was measured at a shear rate of 60/s at 38°C. The viscometer was rinsed with deionized water and wiped clean between samples. The viscosity values were recorded as apparent viscosity in millipascal seconds (mPa·s).

Laboratory Analysis
Bacterial Enumeration.
Approximately 1 g of ileal digesta sample was added to sterile 0.1% peptone (99 mL), massaged for 1 min (Stomacher Lab-Blender 400, Seward Medical, London, UK), and serially diluted in sterile 0.1% peptone (9 mL). For Escherichia coli, dilutions (10⁴ to 10⁸) were plated on petrifilm E. coli/coliform plates (3M Canada Inc., London, Ontario, Canada). Typical colonies were counted after incubation at 35°C for 24 h. Lactobacilli were enumerated by using de Man, Rogosa, Sharpe medium (Becton Dickson & Co.) after incubation at 32°C for 24 h. To determine aerobic and anaerobic spore formers, 5-mL portions of the diluted samples (10¹ to 10⁴) were heated in a thermostatically controlled water bath at 80°C for 15 min. Samples were cooled in an ice bath, plated on Trypticase soy agar (Becton Dickson & Co.), and incubated at 35°C for 24 to 48 h. Anaerobic spore formers were incubated in jars containing anaerobic gas-generating kits (BBL GasPak Plus, Becton Dickson & Co.). All plating was performed in duplicate, and the results were reported as cfu per gram of wet digesta.

Chemical Analyses.
Digesta and fecal samples were freeze-dried and, along with air-dried diet samples (phase II diets only), finely ground in a Smart Grind coffee grinder (Applica Consumer Products Inc., Miami Lakes, FL). Diets, ileal, and fecal samples were analyzed for chromic oxide, DM, CP, and NSP constituent sugars. Chromic oxide was determined by using the procedure of Williams et al. (1962). Dry matter was determined by oven-drying at 105°C for 12 h. The CP (N x 6.25) content was determined with a Leco NS 2000 Nitrogen Analyzer (Leco Corporation, St. Joseph, MI).

Nonstarch polysaccharide concentrations were determined by GLC (component neutral sugars) and by colorimetry (uronic acids), as described by Slominski et al. (2006). Briefly, 100 mg of feed or 50 mg of digesta or feces sample was boiled with 2 mL of dimethylsulfoxide for 1 h and incubated at 45°C overnight with a sodium acetate buffer solution (pH 5.2) containing the starch-degrading enzymes amylase, pullulanase, and amyloglucosidase (Sigma Chemical Co., St. Louis, MO). Ethanol was then added, and the mixture was left for 1 h at room temperature prior to centrifuging. The supernatant was discarded, and the dried residue was dissolved in 1 mL of 12 M sulfuric acid and incubated for 1 h at 35°C. Six milliliters of water and 5 mL of myoinositol (internal standard) solution were added, and the mixture was boiled for 2 h. The resulting hydrolysate was used to determine uronic acids and component sugars. For the component sugars, 1 mL of the hydrolysate was neutralized with 12 M ammonium hydroxide, reduced with sodium borohydride, and acetylated with acetate anhydride in the presence of 1-methylimidazole. Component sugars were separated by using an SP-2340 column and a Varian CP 3380 gas chromatograph (Varian Canada Inc., Mississauga, Ontario, Canada). Uronic acids were determined according to the procedure described by Scott (1979).

Plasma samples were analyzed for PUN with a Nova Stat profile M blood gas and electrolyte analyzer (Nova Biomedical Corporation, Waltham, MA). Ammonia content in the digesta and fecal samples was analyzed as ammonia nitrogen by using the method described by Novozamsky et al. (1974). Briefly, an aliquot (5 g) of digesta or fecal sample was diluted with 0.1 N HCl (1:5, wt/vol), vortexed, and centrifuged for 10 min at 2,000 x g. The supernatant (50 µL) was transferred to a 10-mL test tube, and 1.5 mL of a solution containing 200 mL of 0.05% sodium nitroprusside and 10 mL of 4% EDTA was added and vortexed. A solution containing 10% NaOH (2.5 mL) was then added to the mixture and vortexed. Test tubes containing the resulting mixture were placed in a test tube rack wrapped with black plastic sheets and placed in complete darkness for 30 min, after which the absorbance was read at 630 nm (Milton Roy Spectronic 1001 Plus UV Visible Spectrophotometer, Milton Roy Co., Rochester, NY). Ammonia nitrogen concentrations were determined by calculating the concentrations from a regression equation of the standard curve (range, 25 to 200 mg/L). To obtain the final ammonia concentrations in the sample, values calculated from the standard curve were corrected for dilution.

Organic acids [OA; VFA, lactate, and branched-chain VFA (BCVFA)] were assayed in the ileal, cecal, and colon digesta. Approximately 5 g of the digesta sample was resuspended with 25 mL of 0.1 N HCl (1:5, wt/vol) in a 125-mL conical flask, tightly sealed with Parafilm, and loaded onto a controlled-environment incubator shaker (New Brunswick Scientific Inc., Edison, NJ) set at 180 rpm, at room temperature for 2 h. The resulting digesta fluid was then assayed for OA by using gas chromatography, according to Erwin et al. (1961). Briefly, an aliquot of 2.5 mL of digesta fluid was mixed with 0.5 mL of 25% meta-phosphoric acid in a centrifuge tube and the mixture was frozen overnight. Thawed samples were mixed with 200 µL of 25% NaOH and vortexed, followed by the addition of 320 µL of 0.3 M oxalic acid. The samples were centrifuged for 20 min at 3,000 x g, and the supernatant (2 mL) was transferred to a GLC vial. The OA were determined by using a glass column packed with 80/120 Carbopack B-DA/ 4% Carbowax 20M (Supelco, Bellefonte, PA) in a Varian model 3400 gas chromatograph. The following column conditions were used: initial temperature, 175°C; initial hold time, 20 min; final temperature, 215°C; gradient,20°C/min; carrier gas, helium (flow rate, 24 mL/min). To obtain the final OA concentrations in the sample, gas chromatography readings were corrected for dilution. All analyses were performed in duplicate.

Calculations and Statistical Analyses
The apparent nutrient digestibilities were calculated as described by Opapeju et al. (2006). Bacterial enumeration data were transformed to log₁₀ cfu/g before statistical analysis. Data were analyzed as a completely randomized design with a 2 x 2 factorial treatment arrangement by using the GLM procedure (SAS Inst. Inc., Cary, NC). The sex effect was not significant and was excluded from the model. Subsequently, the final model included the main effects of FS, C, and the associated 2-way interactions. For the performance data (ADG, ADFI, and G:F) and fecal parameters the pen was the experimental unit, but for the other response criteria, pig within diet was the error term. Treatment effects were determined with orthogonal contrasts for a 2 x 2 factorial arrangement. Treatment differences were considered significant at P <0.05 and trends (0.05 >P <0.10) were discussed.

	RESULTS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The analyzed CP and NSP contents for the phase II basal diets are shown in Table 2. The CP values were similar among the diets. The total NSP content in the FS diets was slightly more than in the non-FS diets.

Digesta and Fecal Microbial Activity
Pigs fed the diets with C had a higher ileal lactobacilli count (8.94 vs. 8.25 log₁₀ cfu/g, P = 0.009; Table 4) than pigs fed the diets without C. Pigs fed the FS diets had a lower ileal anaerobic spore former count than pigs fed the non-FS diets (2.69 vs. 3.09 log₁₀ cfu/g, P = 0.033). Escherichia coli and aerobic spore former counts and the lactobacilli to E. coli ratio in pigs were not different (P >0.10) among the diets. However, except for lactobacilli, pigs fed the FS diet supplemented with C had numerically decreased concentrations of all bacteria enumerated compared with pigs fed the other diets.

Flaxseed and C did not interact (P >0.10) to affect VFA, lactate, and total OA concentrations in the ileum (Table 5). Pigs fed the FS diets had decreased ileal concentrations of VFA (5.56 vs. 9.71 mmol/L, P = 0.045), lactate (5.57 vs. 13.3 mmol/L, P = 0.009), and total OA (11 vs. 23 mmol/L, P = 0.006) compared with pigs fed the non-FS diets. Pigs fed the diets with C tended to have greater ileal lactate (12.2 vs. 6.79, mmol/L, P = 0.061) and total OA (20 vs. 14, P = 0.073) concentrations compared with pigs fed the diets without C. There was no treatment effect (P >0.10) on large intestine total OA concentrations. Overall, pigs fed FS and non-FS diets had 10- and 5-fold increase in total cecal OA, respectively, relative to the ileum.

Ileal Digesta Viscosity
An interaction was observed between FS and C in digesta viscosity (P = 0.045; Table 6). Enzyme supplementation increased digesta viscosity in pigs fed the FS diets and slightly decreased viscosity in pigs fed the non-FS diets. Pigs fed the FS-based diets had greater digesta viscosity (3.41 vs. 1.46, P <0.001) than pigs fed the non-FS diets.

There was an interaction (P = 0.004) between FS and C in apparent fecal DM digestibility (Table 6). Enzyme supplementation increased apparent fecal DM digestibility in pigs fed the FS diet and was slightly reduced for pigs fed the non-FS diet. Fecal CP digestibility did not differ among the diets (P > 0.10). Flaxseed and C affected fecal NSP digestibility independently. Except for mannose and galactose, pigs fed the FS diets had greater fecal digestibility of arabinose (68.4 vs. 63.2%, P = 0.022), xylose (67.2 vs. 53.3%, P = 0.001), glucose (55.1 vs. 44.7%, P = 0.001), uronic acid (59.0 vs. 36.5%, P = 0.001), and total NSP (64.0 vs. 54.0%, P = 0.001) compared with pigs fed the non-FS diets. Carbohydrase supplementation had no effect (P > 0.10) on fecal NSP constituent sugars and total NSP digestibility.

	DISCUSSION

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As reviewed by Simon (1998), NSP-depolymerizing carbohydrase enzymes may have several modes of action: partial hydrolysis of NSP, decrease in digesta viscosity, and rupturing of NSP-containing cell walls, thereby making the encapsulated nutrients available for digestion. Other effects include shifts in the population and activity of the microflora as a result of enzyme supplementation (Vahjen et al., 1998). Flaxseed is a rich source of components such as ALA and lignans, which possess broad antimicrobial activity (Pauletti et al., 2000; Kankaanpää et al., 2001). However, the high content of mucilaginous NSP in FS may impair nutrient utilization by increasing digesta viscosity (Bhatty, 1993). We hypothesized that by including a carbohydrase blend in a simple pig starter diet containing 0 or 12% ground FS, more nutrients would be made available to support piglet growth performance and that FS components, as well as enzyme-hydrolysis products from partial NSP breakdown, would affect microbial activity in the gastrointestinal tract, leading to a healthier piglet.

The effect of FS on the overall growth performance in the current study is in agreement with the study reported by Van Kessel et al. (2006). In that study, piglets fed a wheat-soybean meal basal diet or the basal diet with either 5 or 10% ground FS exhibited similar growth performance. However, as demonstrated in wk 3 of the current study and in studies with chickens(Rodríguez et al., 2002), FS can exert depressive effects on the growth performance of young nonruminant animals. This may be due to the presence of various antinutritional factors in this oilseed, such as mucilage, linatine (a vitamin B₆ antagonist), and cyanogenic glycosides (Bhatty, 1993). The concentrations of these antinutritional factors were not determined in the current study or in the study by Van Kessel et al. (2006). It is rather difficult to explain why the depressive effects of FS occurred in the third week and not earlier; however, it takes several days for pigs to recover from weaning effects (Pluske et al., 2002), and it is likely that weaning effects may have confounded the diet effects on growth performance during this period.

In agreement with studies by Högberg and Lindberg (2004) and Zijlstra et al. (2004), enzyme supplementation did not have consistent effects on growth performance variables. This is surprising given that a previous study in this laboratory using the same multicarbohydrase enzyme showed consistent improvement in the growth performance of piglets fed wheat- and soybean-based diets (Omogbenigun et al., 2004). However, in contrast to the current study, pigs in the previous study were given a commercial starter diet for a 1-wk adaptation period immediately after weaning before exposure to the experimental diets. Considering other experimental variations, the lack of an adaptation period in the current study may have contributed to the differences between the 2 studies. Nevertheless, multicarbohydrase supplementation in the FS-based diet improved G:F in wk 3. This indicated a potential for the carbohydrase blend to overcome the depressive effects of FS in the starter diets.

Carbohydrase supplementation reduced PUN in pigs fed the FS diet and not in pigs fed the non-FS diet during wk 1. In addition, pigs fed the FS diets tended to have decreased PUN compared with pigs fed the non-FS diets in wk 1. Plasma urea N has been used as an indicator of AA breakdown as a result of a less than optimal systemic AA supply for protein synthesis (Coma et al., 1996) and of muscle protein breakdown to release AA for synthesizing acute-phase proteins in the liver as a response to immune system activation (Wannemacher, 1977). In this context, McCracken et al. (1995) demonstrated that metabolic responses during the initial days postweaning were due to diet-dependent and diet-independent factors. Thus, we cannot clearly establish what may have caused the observed differences in PUN in wk 1 of the current study. In the third week, however, pigs fed the FS diets had greater PUN concentrations than pigs fed the non-FS diets, which coincided with reduced ADG for pigs receiving this treatment. This suggests that FS may have adversely affected dietary AA availability, leading to a less than optimal systemic AA supply for protein accretion, resulting in increased AA catabolism concomitantly with growth depression. However, the effect of FS on PUN was not detected in wk 4, possibly because the digestive system at this age was capable of overcoming any adverse effect of FS on dietary AA availability.

Lactic acid bacteria, such as lactobacilli and streptococci, dominate microbial populations in the swine small intestine (Pluske et al., 2002). Increasing concentrations of lactate in ileal digesta should therefore reflect an increased population and activity of these microbes (Pluske et al., 2002). In the current study, pigs fed the enzyme-supplemented diets had greater ileal lactate and total OA concentrations as well as increased ileal mannose, arabinose, and xylose digestibility. This indicated that enzyme hydrolysis products containing arabinose, xylose, and mannose sugar residues may have supported lactic acid bacteria activity. Indeed, pigs fed the enzyme-supplemented diets had an increased ileal lactobacilli count compared with pigs fed the unsupplemented diets. These observations support the recent interest in using carbohydrase enzymes to generate carbohydrate fragments capable of supporting beneficial bacterial populations in the gastrointestinal tract (Pluske et al., 2002). Similarly, Högberg and Lindberg (2004) reported an increased molar proportion of lactate in the ileum when piglets were fed cereal-based diets supplemented with xylanase and ß-glucanase. Overall, these findings are of importance relative to the management of weaned pigs, because lactate has been shown to have antibacterial effects on E. coli and Salmonella species (Nout et al., 1989), and lactobacilli have been shown to inhibit adhesion of enterotoxigenic E. coli to the ileal epithelium (Hillman et al., 1995). Although a statistical difference was not observed in the current study, the numerically decreased ileal E. coli count in pigs fed the enzyme-supplemented diets (6.24 vs. 6.72 log₁₀ cfu/g) compared with pigs fed the unsupplemented diets is indicative of suppression of this organism. Furthermore, the reduction in ileal digesta ammonia (as was observed in the enzyme-supplemented non-FS diet) is a further indication of a healthier gut, because a greater ammonia concentration is considered to have negative effects on gut health (Lin and Visek, 1991).

Except for the enzyme-supplemented non-FS diet, the concentrations of lactobacilli in the current study were similar to those reported for ileal digesta of growing (33 kg of BW) pigs fed diets with or without FS (Smith et al., 2004). In that study, ileal concentrations of lactobacilli in pigs fed either a wheat-pea-soybean basal diet or a basal diet plus 20% ground FS were 7.97 and 8.13 log₁₀ cfu/g, respectively. Thus, pigs fed the FS diets exhibited a reduced ileal OA concentration, anaerobic spore former count, and NSP digestibility, which suggest prececal suppression of microbial activity. Furthermore, there was a tendency for an interaction between FS and C in the lactobacilli count such that in the absence of FS, enzyme supplementation increased the lactobacilli count. A reduction in ileal microbial activity in FS-fed pigs (albeit of high viscosity) would appear to contrast with studies in poultry (Langhout et al., 2000) and piglets (McDonald et al., 2001), which showed that high intestinal viscosity is associated with high bacterial activity. However, the current data appear to indicate that other chemical components in the FS may have affected microbial activity in the gastrointestinal tract. For instance, the aforementioned studies used several sources of viscous polysaccharides, such as guar gum, carboxymethylcellulose, pearl barley, and citrus pectin, to elevate intestinal viscosity. These sources are distinctly different from FS, especially in terms of chemical composition. In this context, FS is a rich source of components such as ALA and lignans, which possess broad antimicrobial activity (Pauletti et al., 2000; Kankaanpää et al., 2001). Indeed, ALA from FS has been used in poultry to control the protozoa responsible for coccidiosis (Allen et al., 1997). Similarly, the observed suppression of anaerobic spore formers in the current study is indicative of the potential of FS to reduce intestinal pathogens in piglets, such as Clostridium perfrigens (a common etiological agent in swine enteric diseases and a prominent member of the group of anaerobic spore formers; Moxley and Duhamel, 1999). Clearly, pathways that could suppress microbial activity, especially the pathogenic types, in the ileum of FS-fed piglets require further investigation.

Organic acid and ammonia concentrations were 5- to 10-fold greater in the large intestine than in the ileum and resulted in additional fermentative activities yielding BCVFA (sum of isobutryrate, isovalerate, and valerate). This indicated that there was greater microbial activity and considerable N metabolism in the hindgut. This is not surprising, because it is well established that OM entering the large intestine is subjected to extensive fermentation because of the large reservoir of bacteria (Ewing and Cole, 1994; Pluske et al., 2002). However, the nutritional significance of the hindgut nitrogen metabolism must be considered in the context that the host cannot utilize either bacterial protein or the BCVFA (Dierick and Decuypere, 1996). In contrast to the ileum, dietary treatments did not have significant effects on the large intestine total OA and ammonia concentrations. This suggests that there are greater opportunities to use FS and C to modulate intestinal microbial activity in the ileum than in the large intestine. In addition, the antimicrobial activity of FS may not be as effective in the large intestine, probably because of the large number of bacteria in this section of the gut. In general, our results indicate that lactate was the principal OA in the ileum and VFA dominated in the large intestine, in agreement with earlier findings (Högberg and Lindberg, 2004).

Nyachoti et al. (2006) showed that digesta pH is not well correlated with OA concentration. Subsequently, the observed differences in the ileal OA concentrations were not reflected in ileal pH. It has been suggested that digesta pH is dependent on the individual OA pK values, the proportion of specific OA, and the buffering capacity of dietary nutrients such as protein (Ewing and Cole, 1994). Perhaps the more diversified OA profiles observed in the cecum may explain the decline in cecal pH relative to the ileum. Nonetheless, the gastrointestinal tract pH observed in the current study decreased from the ileum to the cecum, as has been reported for weaned pigs (Högberg and Lindberg, 2004). The colon pH was greater than in the cecum, which coincided with considerably greater ammonia in the former section. The increase in colon pH is an indication that by the time the digesta reached the colon, little fermentable carbohydrate remained and that protein fermentation was occurring and releasing ammonia (which is known to raise pH; Ewing and Cole, 1994). Pigs fed the FS diets had decreased fecal ammonia and pH compared with pigs fed the non-FS diets, indicating disappearance of fermentable protein at the fecal level. Interestingly, pigs fed the C-supplemented diets had greater fecal pH. This may be indicative of increased bacterial protein degradation, more proximal completion of carbohydrate fermentation, or more complete OA absorption (Houdijk et al., 2002).

Viscosity is often measured on the liquid portion of the digesta as separated by centrifugation (Zijlstra et al., 2004). The small intestine is the main site sampled for viscosity measurements in pigs, and the need for dilution does not seem to be a commonly recorded problem because of the fact that there is greater water content in the upper regions of the intestinal tract (McDonald et al., 2001). However, in the current study some samples were too dry to allow for extraction of enough liquid, which necessitated dilution of the intestinal contents to enable comparison among the diets. Similarly, McDonald et al. (2001) diluted dry intestinal samples with water (1:1 wt/vol) to enable viscosity measurement. Because dilution is expected to reduce the actual viscosity (Bedford and Schulze, 1998), the viscosity values in the current study should be viewed as an estimate of apparent viscosity for the purposes of comparison among diets rather than as an absolute quantification.

Simon (1998) has suggested that C are capable of partially degrading soluble NSP into smaller molecular weight polymers and thus decreasing digesta viscosity. However, in the current study, pigs fed the FS diet with C had greater viscosity than pigs fed the FS diet without C, suggesting that the effect of C on NSP-mediated intestinal digesta viscosity may be dependent on the nature of the viscous NSP. In this context, viscosity has been described as a consequence of NSP dissolving in the digestive tract to form high molecular weight viscous aggregates (Bedford and Schulze, 1998). Subsequently, increased viscosity in the presence of C (as was observed in the current study) is an indication of further NSP solubilization. Similarly, an enzyme-mediated increase in digesta viscosity was observed in weaned pigs fed rye-based diets supplemented with pentosanase, and viscosity was associated with enzyme-catalyzed release into solution of insoluble pentosans (Bedford et al., 1992). The soluble NSP in the current study were derived primarily from wheat, barley, and FS, with the canola meal, soybean meal, and peas being minor contributors (Bach Knudsen, 1997). However, their relative contribution to the overall digesta viscosity was such that FS exerted more viscosity than other feedstuffs, as indicated by the high viscosity in FS-based diets. This is not surprising because FS mucilage is a viscous polysaccharide with functional properties similar to those of gum arabic (Rodríguez et al., 2002). Indeed, the FS diets had a slightly greater NSP content.

Pigs fed the FS diets had decreased apparent ileal digestibility of DM and CP, an observation that correlated well with the data discussed earlier demonstrating that FS depressed piglet performance and increased PUN in wk 3. The depressive effect of FS on nutrient digestibility could be attributable to the viscosity of the FS mucilage. Viscosity is considered to be the mechanism by which soluble fiber components, such as ß-glucans, arabinoxylans, gums, mucilage, and pectins, reduce nutrient digestibility by interfering with interactions between digestive enzymes and their substrates (Bedford and Schulze, 1998). This is consistent with the finding that FS diets had the greatest ileal digesta viscosity and the lowest CP and DM digestibilities. The improved DM digestibility in the enzyme-supplemented diets in the current study appears to be due mainly to the increase in NSP digestibility, particularly NSP containing mannose, xylose, and arabinose sugar. The improved fecal DM digestibility in the enzyme-supplemented FS diet could be attributable to the prolonged action of the added enzymes and the presence of micro-organisms in the large intestine.

The loss of 12% total NSP before the terminal ileum of pigs fed the unsupplemented non-FS diet supports suggestions by Gdala et al. (1997) that bacterial activity capable of degrading NSP is present in the upper small intestine. Ileal digestibility of some NSP constituent sugars among the diets and total NSP for the nonenzyme FS diet were negative. Negative ileal digestibility of NSP and constituent sugars has been reported in swine (Graham et al., 1986). To this end, several mechanisms have been advanced for the negative ileal NSP digestibility values: antiperistaltic movements, contaminating endogenous and microbial matter, and phase separation of the digesta and the marker (Graham et al., 1986; Högberg and Lindberg, 2004). Because we did not investigate any of these mechanisms, we cannot definitively identify the probable reasons for the negative ileal digestibility values observed in the current study. However, it is noteworthy that galactose digestibility values are consistent with findings reported by Lien et al. (1997), demonstrating that endogenous mucins accounted for 71 to 82% of galactose in the terminal ileum of growing pigs. However, with the exception of mannose, galactose, and uronic acid, the FS diets had decreased prececal constituent sugars and total NSP digestibility, which coincided with the decreased microbial activity observed in pigs fed these diets. An increase in arabinose, xylose, and mannose digestibility coincided with a tendency for greater OA concentration in ileal digesta of piglets fed the enzyme-supplemented diets.

Total tract NSP digestibility was greater than in the ileum and reflected increased OA and ammonia production in the large intestine. However, in contrast to the ileum, and with the exception of galactose, the FS diets had greater total tract constituent sugars and NSP digestibility than the non-FS diets. This indicated that inclusion of FS in the piglet diet shifted microbial activity from the ileum to the hindgut, which coincided with greater DM digestibility and lower fecal pH and ammonia concentration in pigs fed the FS diets.

In conclusion, the current study demonstrated that FS reduced ileal microbial activity, ileal nutrient digestibility, and piglet performance in wk 3 postweaning. However, it should be noted that diet composition is one of the major factors that can influence nutrient utilization and microbial activity in the gastrointestinal tract, mainly through the contents of antinutritional factors and the nature of the substrate available, respectively. Thus, to clearly delineate effects attributable to FS, feedstuff levels other than FS should be similar among the diets. However, this could not be achieved in the current study because FS, similar to wheat, barley, peas, canola, and soybeans, also supplied energy and nutrients. Indeed, FS contains 20% CP, 41% oil, and 30% dietary fiber on a DM basis (Slominski et al., 2006). However, for pigs fed the FS diets, carbohydrase increased G:F, ileal DM and NSP digestibilities, and lactobacilli and lactate concentrations, and reduced ileal ammonia concentrations. Although these effects were not translated into improved growth performance, it should be pointed out that the current study was conducted in a research facility. Subsequently, the effectiveness of C on piglet intestinal microflora should be evaluated in commercial facilities to fully understand its impact on gut health and performance. Furthermore, the prospects for using C to hydrolyze FS NSP, thus allowing inclusion of FS in the starter diets, need to be evaluated in relation to the antimicrobial properties of the FS fractions.

	Footnotes

 
¹ Funding for this project from Canadian Bio-Systems Inc., Manitoba Pork Council, and Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. E. Kiarie was in receipt of the University of Manitoba graduate fellowship. Special thanks to R. Stuski and G. H. Crow for help with animal care and statistical analysis, respectively.

² Presented in part at the 26th World Congress and Exhibition of the International Society for Fat Research, Prague, Czech Republic, September 25–28, 2005, and at the 2006 ADSA-ASAS joint meeting, Minneapolis, Minnesota, July 9–13, 2006.

³ Corresponding author: umkiarie{at}cc.umanitoba.ca

Received for publication July 19, 2007. Accepted for publication July 11, 2007.

	LITERATURE CITED

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 


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