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ANIMAL NUTRITION |
* Departamento de Producción Animal, Universidad Politécnica de Madrid, E.T.S. Ingenieros Agrónomos, 28040 Madrid, Spain;
and
Servicio de Inmunología del Hospital Ramón y Cajal, 28034 Madrid, Spain; and
CreSA (UAB-IRTA), Bellaterra, 08193, Barcelona, Spain
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.38). Rabbits fed the B-AP and AH diets had a reduced cecal frequency of detection compatible with Campylobacter spp. (20.3 vs. 37.8, P = 0.074), and Clostridium perfringens (4.3 vs. 17.6%, P = 0.047), compared with the OH diet group. Moreover, the mortality rates decreased from 14.4 (OH diet) to 5.1% (B-AP diet) with the increased presence of soluble fiber in the diet. In conclusion, increased levels of dietary soluble fiber improve mucosal integrity and functionality.
Key Words: immune response • intestinal microbiota • mucosa integrity • rabbit • soluble fiber
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). This has increased the demand for nutritional strategies to reduce their incidence and the use of antibiotics, particularly in the postweaning period. At weaning, rabbits have a greater risk of disease because the colonization of the gut by microbiota (Gouet and Fonty, 1979; Padilha et al., 1995) and the development of gut-associated lymphoid tissue is not completed at this stage (Dasso et al., 2000; Lanning et al., 2000; Brown et al., 2006). Furthermore, a reduction of both the villous/crypt height ratio and the activity of the jejunal brush border enzymes (sucrase and maltase) has been observed just after weaning (Gutiérrez et al., 2002), and this may indicate an impairment of mucosal functional capacity (Henning, 1985; Hampson and Kidder, 1986; Tang et al., 1999).
The dietary fraction most related to digestive troubles in rabbits is insoluble fiber, which is the most important determinant of rate of passage and growth of microbes (De Blas et al., 1999; García et al., 2000; Gidenne, 2003). Furthermore, at the same concentration of dietary fiber, the dietary inclusion of soluble and fermentable fiber decreases the pH and increases the total cecal concentration of VFA and the total mean retention time (García et al., 1993; Carabaño et al., 1997). Previous studies in rabbits have also shown that pectin compared with highly lignified fibers improved morphology of the intestinal mucosa and increased the activity of intestinal cells (Chiou et al., 1994). However, the implications of these changes in the intestinal microbiota and their relationship to intestinal health in rabbits are currently unknown. The objective of this study was to analyze the effects of dietary soluble fiber on gut barrier function in rabbits during the postweaning period.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This study was approved by the Committee of Ethics of the Departamento de Producción Animal of the Universidad Politécnica de Madrid. All animals were handled according to the principles of animal care published by Spanish Royal Decree 1201/2005 (BOE, 2005).
Crossbred (New Zealand White x Californian) rabbits obtained from a farm affected by epizootic rabbit enteropathy (ERE) were used in all of the experiments. Each of the animals appeared healthy at the beginning of the study, and no mortality was observed during the 10-d period from 25 to 35 d old. Animals were individually housed in flat-deck cages of 600 x 250 x 330 mm, and housing conditions were controlled during the whole experimental period as follows: a 12-h light:dark cycle was established and temperature conditions were maintained between 15 and 24°C.
Experimental Diets
Three starter diets with different soluble fiber contents (measured as neutral detergent-soluble fiber) were formulated (Table 1). A control diet (AH) containing 103 g/kg (DM basis) of soluble fiber was established by including alfalfa hay as the main source of fiber. A diet containing greater soluble fiber (B-AP) was formulated by replacing half of the alfalfa hay used in the AH diet with a mixture of beet and apple pulp (75:25), resulting in a soluble fiber content of 131 g/ kg of DM. A third lower soluble fiber diet (OH) was developed by substituting half of the alfalfa hay with a mixture of oat hulls and a soybean concentrate (88:12, wt/wt; Soycomil P-economy, Loders Croklaan, Wormerveer, the Netherlands) and contained 79 g of soluble fiber/kg of DM.
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; Gutiérrez et al., 2002). They also contained similar concentrations of total dietary fiber (NDF + soluble fiber), starch, and CP (Table 1
). To determine the apparent ileal digestibility, 5 g of DM/kg of alfalfa hay labeled with Yb2O3 was included in all diets, and analyzed according to the procedures described by García et al. (1999). Rabbits had ad libitum access to feed and water, and a mixture of 100 ppm of apramicine sulfate and 120 ppm of tylosine tartrate was supplied by water to all animals from 25 to 35 d of age. Before the experimental period (before weaning), both weanling rabbits and their mothers were given ad libitum access to a commercial feed during lactation (Cunilactal, NANTA, S.A., Spain) containing (g/kg of DM); 182 CP, 359 NDF, 45 ADL, 161 starch, and 53 ether extract. Experiment 1: Determination of Ileal Digestibility, Mucosa Morphology, and Sucrase Activity
Eighty-four weanling rabbits of both sexes, 25 d in age, and a BW of 535 ± 8.6 g were blocked by litter and randomly assigned to the 3 experimental diets (28 rabbits/diet) to determine the apparent ileal digestibility of DM, CP, and starch. After a 10-d experimental period, 35-d-old animals with a BW of 1,034 ± 14.9 g were killed between 1900 and 2100 h to minimize the influence of cecotrophy. The caudal 20 cm of the ileum in each case was then excised, emptied, and the digesta were frozen in dry ice. For the chemical analysis of the ileal digesta, samples were freeze-dried and ground. Due to the small amounts of sample, ileal digesta from 3 to 5 rabbits for each treatment were pooled, resulting in 7 samples/treatment. The ileal digestibility of DM, starch, and CP was determined by the dilution technique using ytterbium as a marker as follows:
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The ileal flow of DM, starch, and CP was calculated by multiplying the apparent ileal digestibilities obtained for each pool of samples and the average daily intake of DM, starch, and CP recorded for the corresponding animals.
Two 6-cm samples were excised from the middle part of the jejunum for 30 of the killed rabbits. One of these samples from each animal was then flushed with saline solution, frozen in dry ice, and immediately stored at –20°C to determine sucrase activity (10/treatment). The second of the 6-cm samples (taken in 8 rabbits/treatment) was rinsed with KCl solution and placed into a 10%, buffered, neutral formaldehyde solution (pH 7.2 to 7.4) to evaluate the mucosal morphology.
In a parallel experiment, samples from a control group of 19 suckling rabbits (35 d old) with a BW of 876 ± 17.0 g were processed in the same way to assess their mucosal morphologies. The jejunal samples used in these analyses were gradually dehydrated in an ethanol series (50 to 100%), embedded in paraffin, sectioned at 6 µm and stained with hematoxylin and eosin (Armed Forces Institute of Pathology, 1968). Three to five slides containing jejunal cross sections were prepared for each sample and were viewed at 40x magnification using an Olympus BX-40 light microscope. Images were digitally captured for later analysis using Soft software version 3.2 C4040Z (Soft Imaging System, Olympus, GmbH, Hamburg, Germany). The villous heights and crypt depths were determined according to the procedure described by Hampson (1986), and an average of the measurements for 3 independent cross-sectional jejunal samples (for which at least 10 measurements were done in each case) was obtained for each animal.
Experiment 2: Characterization of Lamina Propria Lymphocytes
Thirty-six weanling rabbits of both sexes (12/treatment), of 25 d in age, and weighing 529 ± 33 g of BW, were blocked by litter and randomly assigned to the 3 experimental diets. Having reached 35 d in age and a BW of 972 ± 46 g, the animals were euthanized and 1-mm samples were excised from the duodenum just caudal to the pancreatic flexure and processed immediately. The isolation of lymphocytes in the lamina propria was conducted according to the procedure previously described by Eiras et al. (1998). Duodenal samples were longitudinally opened and incubated in 5 mL of a 10% RPMI/fetal bovine serum solution containing 50 µL of collagenase (1.5 mg/mL; Cymbus Bioscience, Eastleigh, UK). For the phenotypic characterization of lamina propria lymphocytes and measurement of their activation grade, commercial monoclonal antibodies were used (Cymbus Biotechnology, Eastleigh, UK). Anti-rabbit CD45 was used for the localization of all types of lymphocytes, CD5 antibodies were employed to distinguish T from B lymphocytes, CD25 antibodies were used to identify T lymphocytes that express the interleukin-2 (IL-2) receptor, CD4 antibody was used as a marker for T helper lymphocytes, and CD8 antibody was used to identify suppressor T lymphocytes. Data analysis was performed using flow cytometry (Tricolor flux in FACScan standard, Becton Dickinson, S. Agustín de Guadalix, Spain).
Experiment 3: Characterization of Intestinal Microbiota and Determination of Mortality Rate
One hundred eleven 25-d-old weanling rabbits of both sexes weighing 423 ± 11 g of BW were blocked by litter and randomly assigned to the 3 experimental diets. The animals were euthanized at 35 d between 1900 and 2100 h, and approximately 1 g of both ileal (from 18 to 22 rabbits/treatment) and cecal samples (from 33 to 37 rabbits/treatment) were collected in sterile plastic tubes containing 3 mL of 98% molecular biology grade ethanol. The collected samples were maintained at 4°C until use. Several rabbits had no ileal content, and the number of ileal samples was therefore lower than the cecal sample number.
Another 354 rabbits, weighing 485 ± 6 g, were blocked by litter and assigned at random to the 3 dietary treatments (118 animals/diet). Each experimental diet was offered ad libitum for 2 wk after weaning (at 25 d of age). After this period, all of these animals were given a commercial feed (Cuniunic, Nanta, S.A.), containing (g/kg as fed): 160 CP, 345 NDF, 50 ADL, and 144 starch. Mortality was recorded from the time of weaning until 63 d of age.
Chemical Analysis
Procedures of the AOAC (2000) were used to determine the concentrations of DM (934.01), ash (967.05), CP (968.06), ether extract (920.39), and starch (amyloglucosidase-
-amylase method, 996.11). Dietary NDF, ADF, and ADL were determined sequentially using the filter bag system (Ankom Technology, New York, NY) according to the methods of Mertens (2002), the AOAC (2000; procedure 973.187), and Van Soest et al. (1991), respectively. Soluble dietary fiber was analyzed as a neutral detergent-soluble fiber according to Hall et al. (1997), and included fructans, galactans, ß-glucans, and pectic substances.
For the determination of sucrase activity (EC 3.2.1.48), jejunal samples were thawed, and the mucosa was scraped using a blunt spatula and placed into vials containing distilled water to form a 1:20 (wt/vol) mucosal homogenate. This was followed by homogenization for 30 s in a Polytron (model RE 16, Janke & Kunkel, IKA-Labortechnik, Staufen, Germany). The supernatant was decanted and stored in vials at –20°C. Sucrase activity (EC 3.2.1.48) was determined using sucrose (Sigma, St. Louis, MO) as a substrate. The concentrations of glucose liberated at 37°C for 30 min were determined by UV using the glucose-6-phosphate dehydrogenase (EC 1.1.1.49)-hexoquinase (EC 2.7.1.1) assay (Boehringer-Mannheim Biochemica, Mannheim, Germany; Schmidt, 1973). The protein contents of the mucosal homogenates were determined using a commercial kit (Sigma Procedure No. P-5656, Sigma-Aldrich, Tres Cantos, Spain), based on Lowry’s method as modified by Peterson (1977).
Microbiota biodiversity and detection frequencies were determined by 16S rRNA RFLP according to the following procedure: 400 mg of gut contents were processed for total DNA extraction using the QIAamp DNA Stool Mini Kit system (Qiagen Inc., Chatsworth, CA) according to the manufacturer’s instructions, with additional lysozyme and proteinase K steps. The purified DNA was maintained at –20°C until use. The primers 5'-CTACGGGAGGCAGCAGT-3' and 5'-CCGTCWATT-CMTTTGAGTTT-3', corresponding to regions I and II of the 16S rRNA gene (Lane, 1991), were used to amplify a 500- to 600-bp product. Amplifications were performed in a final volume of 50 µL using a PCR-Master Mix (Applied Biosystems) containing 1.25 IU of Taq polymerase, 50 ng of DNA template, 0.2 µM of each primer, and the following cycling conditions: 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 45°C for 1 min, and 72°C for 1 m 15 s. The last extension cycle was continued for 5 min. Aliquots of the amplified DNA fragments were digested, in separated tubes, with Alu I, Rsa I, Hpa II, Sau 3A I, or Cfo I restriction endonucleases (Sigma-Aldrich). The endonuclease fragments were resolved in 2% agarose gels at 150 V for 60 min, and DNA bands were visualized using a UV Chemigenious Image System at a 4.63-s exposure (SynGene) using GeneSnap software (SynGene, Cambridge, UK).
Using the relative sizes of the RFLP bands obtained in our analyses and the information stored in the "SSU_Una.gb" file from the Ribosomal Database Project (Maidak et al., 1997), we could identify the bacterial genus or species compatible with the obtained RFLP profile using specific software developed in our Institute. From each animal, a biodiversity degree, defined as the number of 16S rDNA (genes that codify the rRNA) sequences deposited in the Ribosomal Database Project, that was compatible with the RFLP profile obtained from the total DNA extracted from the gut samples was recorded (Andrés-Elias et al., 2007). In addition, the frequency of detection of each bacterial strain examined was used to define the presence or absence of a specific genus or bacterial species for each animal.
Statistical Analysis
The results obtained in this study for apparent ileal digestibility, ADFI, ileal flow, mucosa morphology traits, sucrase activity, intestinal microbiota, and mortality rates were analyzed as a completely randomized block, with the type of diet used as the main source of variation and the litter as a block effect using the GLM procedure (SAS Inst. Inc., Cary, NC). Weaning weight was included as a linear covariate. Means were compared using a protected t-test, and differences were considered significant at P < 0.05.
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RESULTS |
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Daily feed intake, and the apparent ileal digestibility and ileal flow of DM did not differ among the 3 experimental diets tested (P 0.16), which measured on average 79.7 g of DM, 46.8% and 42.1 g of DM/d, respectively(Table 2). Starch ileal digestibility increased (P = 0.002) with dietary soluble fiber by 3.9% between the extreme diets, which resulted in a 58% reduction of ileal starch flow (P = 0.001) in rabbits fed B-AP compared with those given the OH diet. In contrast, the apparent ileal digestibility of CP showed a tendency for a 15% decrease with soluble fiber (P = 0.074), with no effects on the ileal flow of CP.
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). In addition, the animals who received the greatest level of soluble fiber (B-AP diet) showed a 22% reduction (P = 0.001) in crypt depth compared with those on both the AH and OH diets, which did not differ. Accordingly, animals given the B-AP diet showed the highest ratio villous height/ crypt depth (P = 0.001), whereas no differences were detected between animals who received the AH and OH diets. Suckling rabbits of the same age showed a shorter villus compared with animals fed with the greatest levels of soluble fiber (B-AP diet), and these values were similar and greater than those obtained for the AH and OH diets, respectively. Suckling rabbits also showed the same crypt depths as the animals who had been fed with the B-AP diet, and these were shorter than the AH and OH animals. As a result, the villous height/ crypt depth ratio for suckling rabbits was lower compared with animals given the B-AP diet, but higher than the AH and OH groups.
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) had increased (P = 0.019) in rabbits on the B-AP diet, but no differences were evident between the AH and OH diet groups. No differences were observed between the protein contents of the jejunal mucosa in any of the diet groups, and this value was 70.7 mg/g of tissue on average (data not shown). Experiment 2: Characterization of Lamina Propria Lymphocytes
Differences in the levels of soluble dietary fiber did not affect the percentage of lymphocytes in the lamina propria, which averaged 6.22% of the total cells (Table 4). In addition, no effects of the different diets upon the proportions of T lymphocytes (CD5+), B lymphocytes (CD5–), T helper lymphocytes (CD4+), or CD4+CD8+ lymphocytes were detectable, which measured 75.6, 24.4, 31.7, and 23.7%, respectively. However, the proportion of T lymphocytes harboring an activated IL-2 receptor (CD25+) was decreased (P = 0.029) in animals fed with the lowest level of soluble fiber (OH diet). The rabbits in the OH group also tended (P = 0.057) to show a lower activation of T lymphocytes (CD5+CD25+). In addition, the proportion of CD8+ lymphocytes tended to increase (P = 0.074) with soluble-fiber reduction.
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The dietary treatments did not affect the biodiversity of the ileal microbiota, which showed an average of 591 sequences recognized by SSU Unal.gb (Ribosomal Database Project), and an ileal detection frequency compatible with Bacteroides spp., Bacteroides fragilis, Clostridium spp., Clostridium difficile, Escherichia coli, Propionibacterium spp., and Ruminococus spp. that averaged 67.1, 7.03, 85.0, 5.37, 6.55, 28.2, and 29.0%, respectively (Table 5). However, the ileal frequency profile compatible with Clostridium perfringens, Butirivibrio fibrosolvens, and Campylobacter spp. tended to decrease in animals given the highest levels of soluble fiber (P < 0.079).
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Mortality was affected by diet (SEM = 2.7; P = 0.050) and decreased from 14.4% (OH diet) to 5.1% (B-AP diet) with increased dietary soluble fiber. Rabbits fed the AH diet showed an intermediate result (8.5%). Most of the dead rabbits showed symptoms of borborygmus (stomach rumbling), the presence of mucus in the colon and ileum, and cecal impaction, indicating that the ERE was the underlying cause of death (Peréz de Rozas et al., 2005; Marlier et al., 2006).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Licois et al., 2005; Peréz de Rozas et al., 2005). The etiology of ERE has not yet been elucidated, but proper medication does reduce the incidence of death. Therefore, most farmed rabbits are medicated under field conditions. At present, ERE is the main cause of rabbit mortality in our research facility and the present experiments were carried out under conditions in which this disease can occur, similar to commercial farms.
The intestinal barrier plays a key role in the protection of rabbits and other animals against pathogens because it prevents the colonization and the translocation of bacteria and toxins. Several criteria have been proposed to characterize mucosal integrity. The villous height/crypt ratio and the activities of one or more mucosal enzymes (sucraseisomaltase, aminopeptidase, or alkaline phosphatase) are the most widely used of these to estimate both mucosal integrity and enterocyte maturity in nonruminants (Van der Kils and Jansman, 2002). In our current study, an increase in dietary soluble fiber increased villous height of the jejunal mucosa in weaned rabbits. In addition, rabbits fed the greatest levels of soluble fiber (the BA-P diet) showed a reduction in their crypt depth and an increase in the villous height/crypt depth ratio. Therefore, our current data are in agreement with the earlier findings of Chiou et al. (1994), which showed that the substitution of lignin with pectin improved villous height/crypt depth ratio in purified diets.
In addition to the improvements in the mucosal morphology with increasing levels of soluble fiber, rabbits given the BA-P diet also showed the greatest sucrase activities and ileal starch digestibility. These findings are consistent with the studies of Chun et al. (1989) and Lizardo et al. (1997) who reported an increase in brush border enzyme activity with increasing levels of soluble fiber in rats and pigs, respectively.
The beneficial effects of soluble fiber were further evident when 35-d-old rabbits in the B-AP diet group were compared with suckling rabbits of the same age and are in agreement with Gallois et al. (2005). We previously showed also that low soluble fiber diets are associated with the impairment of the mucosal morphology in weaned rabbits (Gutiérrez et al., 2002). These results indicate the potential benefits of formulating specific postweaning diets for rabbits based on the inclusion of moderate levels of soluble fiber.
Mucosal atrophy and decreased enterocyte functionality were observed in the animals fed with a low soluble fiber diet, and these might cause an enhanced permeability of the mucosa to pathogens. We also observed in our present analyses that there was a trend to increase the cellular immune response (CD8+ lymphocytes) associated with lower levels of soluble fiber. A similar immune response was observed in an earlier study of rats and dogs given different levels of soluble fiber (Lim et al., 1997; Field et al., 1999). We thus hypothesize that dietary soluble fiber has a protective effect upon the mucosa that favors an immune response. Furthermore, this possibility and the improvement of mucosa functionality is consistent also with our observation that there is a reduction in the mortality rate for rabbits fed with moderate levels of soluble fiber. However, further investigations are necessary to clarify the role of soluble fiber on Th1 cytokines as previously suggested Field et al. (1999). We have observed an effect of diet on IL-2 production (CD25+ and CD5+CD25+), but we did not detect a clear relationship between soluble fiber and IL-2.
The influence of the dietary levels of soluble fiber on mucosal integrity and on the immune response might also be mediated by their effects on the proliferation of microbiota. In our current study, rabbits fed with the highest levels of soluble fiber tended to show a reduction in the ileal frequency of detection that is compatible with Clostridium perfringens. In the cecum of rabbits fed with normal and moderate soluble fiber levels (AH and B-AP diets) also decreased the frequency of detection that is compatible with Clostridium perfringens. In fact, recent reports suggest that Clostridium perfringens might be implicated in the incidence of ERE (Peréz de Rozas et al., 2005; Marlier et al., 2006). The negative effects of these bacteria are exacerbated by existing intestinal damage, as demonstrated in broilers affected by necrotic enteritis (Van Immerseel et al., 2004). Differences observed among diets that differ in the substrate availability at the end of the ileum might also favor the proliferation of some pathogens or commensal bacteria. An increase in CP flow at the ileum has been shown to favor C. perfringens proliferation (Wilkie et al., 2005; Chamorro et al., 2007), but in our study, no differences were detectable in the ileal CP flow. The decrease in the ileal flow of starch observed in rabbits fed the B-AP diet might have reduced the sporulation and toxin formation of Clostridium perfringens (Labbe and Duncan, 1975). However, other factors might also be implicated because the differences in ileal starch flow between animals fed AH and B-AP diets were not observed in the presence of Clostridium perfringens. The enterotoxin produced by Clostridium perfringens might also induce cellular damage of the intestinal mucosa and alter its membrane permeability (McClane, 2001; Berkes et al., 2003). In our present analyses, a decrease of the level of soluble fiber also tended to increase the frequency of detection of Campylobacter spp.
Commensal bacteria are poorly described in rabbits but some genera (e.g., Bacteroides and Clostridium) seem to be predominant (Gouet and Fonty, 1979). The main microbial enzyme activity in the ileum and cecum corresponds to pectin-degrading enzymes (Marounek et al., 1995). Furthermore, bacteria belonging to the genus Bacteroides are the principal pectinolytic organisms in the rabbit cecum (Sirotek et al., 2003). However, no clear relationship has been observed between soluble fiber and the presence of some fibrolytic bacteria that might act as a probiotic (Bacteroides spp., Butirivibrio fibrosolvens, Propionibacterium spp., and Ruminococus spp.; Michel et al., 2005; Ohkawara et al., 2005).
In conclusion, an increase in soluble dietary fiber in rabbits improves mucosal integrity and functionality. However, the changes detected in the immune response in lamina propria and in the profile of intestinal microbiota are not consistent with the changes in soluble fiber.
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Footnotes |
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2 Corresponding author: rosa.carabano{at}upm.es
Received for publication November 27, 2006. Accepted for publication August 16, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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