Relationship between dietary-induced changes in intestinal commensal microflora and duodenojejunal myoelectric activity monitored by radiotelemetry in the rat in vivo

Authors


Corresponding author V. Lesniewska: NICHE, School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Co. Londonderry, BT52 1SA, UK. Email: v.lesniewska@ulster.ac.uk

Abstract

Interdigestive intestinal motility, and especially phase III of the migrating myoelectric/motor complex (MMC), is responsible for intestinal clearance and plays an important role in prevention of bacterial overgrowth and translocation in the gut. Yet previous results from gnotobiotic rats have shown that intestinal microflora can themselves affect the characteristics of the myoelectric activity of the gut during the interdigestive state. Given that the composition of the intestinal microflora can be altered by dietary manipulations, we investigated the effect of supplementation of the diet with synbiotics on intestinal microflora structure and the duodenojejunal myoelectric activity in the rat. To reduce animal distress caused by restraint and handling, which can itself affect GI motility, we applied radiotelemetry for duodenojejunal EMG recordings in conscious, freely moving rats. Thirty 16-month-old Spraque–Dawley rats were used. The diet for 15 rats (E group) was supplemented with chicory inulin, Lactobacillus rhamnosus and Bifidobacterium lactis. The remaining 15 rats were fed control diet without supplements (C group). Three rats from each group were implanted with three bipolar electrodes positioned at 2, 14 and 28 cm distal to the pylorus. After recovery, two 6 h recordings of duodenojejunal EMG were carried out on each operated rat. Subsequently, group C rats received feed supplements and group E rats received only control diet for 1 week, and an additional two 6 h recordings were carried out on each of these rats. Non-operated C and E rats were killed and samples of GI tract were collected for microbiological analyses. Supplementation of the diet with the pro- and prebiotics mixture increased the number of bifidobacteria, whereas it decreased the number of enterobacteria in jejunum, ileum, caecum and colon. In both caecum and colon, the dietary supplementation increased the number of total anaerobes and lactobacilli. Treatment with synbiotics increased occurrence of phase III of the MMC at all three levels of the small intestine. The propagation velocity of phase III in the whole recording segment was also increased from 3.7 ± 0.2 to 4.4 ± 0.2 cm min−1 by dietary treatment. Treatment with synbiotics increased the frequency of response potentials of the propagated phase III of the MMC at both levels of the jejunum, but not in the duodenum. In both parts of the jejunum, the supplementation of the diet significantly decreased the duration of phase II of the MMC, while it did not change the duration of phase I and phase III. Using the telemetry technique it was demonstrated that changes in the gastrointestinal microflora exhibited an intestinal motility response and, more importantly, that such changes can be initiated by the addition of synbiotics to the diet.

The small intestine contains a bacterial population that can become opportunistic when physiological defence mechanisms fail. Translocation of gut bacteria and their toxins across the mucosal barrier is considered to be an early phenomenon of endotoxemia, sepsis and multiple organ failure (Yao et al. 1995; Luyer et al. 2004). The physiological mechanisms that regulate the intestinal microflora therefore play an important defence role against gut-derived infectious threats. The interdigestive intestinal motility pattern, and specifically the regular occurrence of phase III of migrating myoelectric/motor complex (MMC) is considered to be one of the physiological mechanisms that prevents bacterial overgrowth (Vantrappen et al. 1977; Scot & Cahall, 1982) and translocation (Nieuwenhuijs et al. 1998) in the gut. Furthermore, it was shown in human subjects that the degree of bacterial colonization of the gut is not only related to the presence/absence of the MMC, but also to MMC intensity (Soudah et al. 1991).

More recent studies on the relationship between intestinal motility and intestinal microflora demonstrated a further complexity. It has been shown that inoculation of germ-free (GF) rats with conventional microflora shortened the duration of MMC cycles by over 50% and that the phase III migrated further aborally (Husebye et al. 1994). More importantly, the same researchers showed that the characteristics of MMC activity were different depending on the species of bacteria used to inoculate GF rats. In rats inoculated with Lactobacillus acidophilus A10 and Bifidobacterium bifidum B11 the duration of MMC cycles was decreased compared to that observed in control GF animals, whereas in rats inoculated with Micrococcus luteus and Escherichia coli X7 the duration of the MMC cycles was increased compared to that observed in control GF rats (Husebye et al. 2001). The results of the above studies from GF animals indicate that the gut motility response can depend on the composition of the intestinal microflora. However, the effect of the changes in the composition of the microflora that can be observed with increasing age in animals and humans (Benno et al. 1992; Woodmansey et al. 2004) or induced by dietary manipulation (Apajalahti et al. 2001; Naughton et al. 2001; Mikkelsen et al. 2004; Hedemann et al. 2005) on the intestinal motility is unknown.

There is no available method for in vitro evaluation of intestinal MMC, and to date research relies on direct or indirect in vivo methods of motility measurement. Hitherto, these methods have required the restraint of experimental animals. However, intestinal motility is significantly inhibited by experimentally applied restraint in the rat (Brown & Groves, 1966; Tsukada et al. 2000). Therefore, we propose a radiotelemetry method for the evaluation of the intestinal myoelectric activity in freely moving rats as method for studying the effect of diet and the bacteria on gastrointestinal motility. Radiotelemetry combines miniature sensors and transmitters to detect biological signals in animals and to broadcast signals to a remote receiver without a need for animal handling and restraint. Therefore, an in vivo model system which allows the evaluation of the intestinal motility without the need for restraint (and the resultant stress) provides a distinct scientific advantage, as was previously shown in GI tract research in pigs (Gacsalyi et al. 2000).

In this study, the radiotelemetry method was implemented to investigate whether dietary-induced changes in the intestinal microflora population affect small intestinal motility in the conventional state. Sixteen-month-old rats with normal bacteria were fed a diet supplemented with a mixture of prebiotic, an oligofructose-enriched inulin in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis, and its effect on the gut motility was evaluated.

Methods

Bacterial strains

Lactobacillus delbrueckii ssp. rhamnosus strain GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) were provided by Valio (Helsinki, Finland) and purchased from Chr. Hansen (Horsholm, Denmark), respectively. They were supplied as a freeze-dried powder that contained 1 × 1013 to 2 × 1013 CFU g−1 in the case of LGG and ∼5 × 1011 CFU g−1 in the case of Bb12. For the purpose of the experiment, the concentration of LGG and Bb12 of different batches was evaluated by dissolving 0.1 g of the powder in 10 ml of Maximum Recovery Diluent (MRD; Oxoid, Basingstoke, UK). Serial dilutions were prepared using the same diluent and plating them on Rogosa agar (Oxoid) containing glacial acetic acid (1.32 ml l−1) for lactobacilli and a selective media for bifidobacteria [BSM; Rogosa agar containing 0.05% (w/v) cysteine hydrochloride (Sigma, Gillingham, UK) and 50 mg l−1 of Mupirocin (Oxoid)] as described by Leuschner et al. (2003). Plates were incubated in an anaerobic cabinet at 37°C: Rogosa agar plates for 48 h and BSM plates for 72 h. When the two strains were plated together (Wilkins Chalgren agar; Oxoid) they were easily recognizable as LGG forms large colonies, whereas those of Bb12 are pinpoint in size.

Animals and diets

All management and experimental procedures conducted during this study were performed in accordance with the European Community regulations concerning the welfare of experimental animals.

Control diet (AIN-93M synthetic meal rodent maintenance diet; Reeves et al. 1993) was purchased from Lillico, Betchworth, UK. Raftilose® Synergy1 (briefly Synergy1) food additive, an oligofructose-enriched inulin, was provided by ORAFTI (Tienen, Belgium). Synergy1 is a 1:1 mixture of long-chain and short-chain fractions of inulin, a β(2-1)-fructan extracted from chicory roots (Cichorium intybus) as described elsewhere (Femia et al. 2002).

Thirty conventional Sprague–Dawley male rats, weighing 554 ± 7 g, were housed individually and kept in a temperature-controlled environment (22 ± 2°C) with a 12 h light–dark cycle. All rats were fed with AIN-93M rodent maintenance (control) diet at 4 g (100 g live body weight)−1 24 h−1. The daily amount of the diet per gram of rat was estimated based on the average initial body weight of the rats measured every week. After 2 weeks of adaptation, the diet of 15 randomly chosen rats (group E) was supplemented with 8% of Synergy1 and with LGG and Bb12 to provide 2.2 × 109 CFU of each strain per gram of the diet. The required amount of Synergy1, LGG and Bb12 was mixed with the daily diet allowance for each animal. The remaining rats (group C) were fed AIN-93M without supplements. All rats had free access to distilled water at all times. Food consumption was monitored daily and body weight was monitored weekly.

Experimental protocols

An overview of the experimental protocols is shown in Fig. 1. Seven days from the beginning of the treatment 12 rats were killed (6 animals for each feeding group), and various regions of gastrointestinal (GI) tract were sampled for microbiological analyses. At the same time point, an additional six rats (3 randomly chosen animals from each feeding group) were surgically modified for intestinal smooth muscle electromyography (EMG) in vivo. After an additional 14 days of the experiment (21 days from the beginning of the treatment) 12 rats were killed and sampled for microbiological analyses. At the same time point, two 6 h recordings of intestinal EMG were carried out on each operated rat. Subsequently, operated rats from C group received feed supplements and rats from E group received only control diet for 1 week, since it has been shown elsewhere that different probiotic strains colonize rat gastrointestinal tract after 7 days of oral administration (Femia et al. 2002) and persist for up to 5 days following cessation of the oral administration (Locascio et al. 2001; Weese et al. 2003). An additional two 6 h recordings were carried out on each operated rat 1 week after the dietary change.

Figure 1.

An overview of the experimental protocol
Four 6 h EMG recordings were carried out on each of the operated rats, two recordings before and two after the change of the diet.

Surgery, telemetry system and EMG recordings

Under general anaesthesia of induced and maintained with 2% (v/v) of isoflurane (Baxter A/S, Allerød, Denmark) in a gas mixture of O2 and NO2 (at the ratio of 2 : 3), rats were implanted with a three-channel radiotelemetry transmitter (TL10M3-F50-EEE, DSI, St Paul, MN, USA), equipped with three bipolar electrodes, as previously described (Lesniewska et al. 2004). The animals were given a single dose of 0.03 mg kg−1 buprenorphine (Temgesic, Schering-Plough, Brussels, Belgium) subcutaneously prior to the surgery and twice a day during the first 2 days after the surgery, for analgesia. The recovery from surgery was uneventful.

The EMG signals from the radiotelemetry transmitter were collected by a receiver (RPC1, DSI) placed under the animal's cage. The receiver was coupled to the analog output (DL10, DSI). Each of three signal channels was then amplified (BioAmp, ADInstruments, Melbourne, Australia), recorded and filtered with an analog-to-digital Mac-based data recording system (PowerLab 8e, ADInstruments), as previously described (Lesniewska et al. 2000). An example of the EMG recording obtained using radiotelemetry in a rat is shown in Fig. 2.

Figure 2.

EMG recording of the myoelectric activity of duodenum (electrode 1) and jejunum (electrodes 2 and 3) obtained using radiotelemetry in a rat

The following temporal and spatial characteristics of the EMG recordings were determined: (i) total duration of MMC cycles (min); (ii) duration of particular phases (I, II and III) of the MMC cycle (min); (iii) the frequency of the response potentials (number of spikes per minute during phase II and III of the MMC); (iv) the propagation velocity of phase III in the recorded intestinal segment (cm min−1); and (v) the aboral propagation of phase III. The occurrence of phase III was calculated for each respective electrode site as the percentage of the number of phases III at each respective electrode site in relation to all occurring phases III in the recording segments. The propagation ratio (R), as defined by Husebye et al. (1994), was used to determine an aboral propagation of phase III for the whole recorded intestinal segment. R was calculated as follows: R=[T/(n0− 1)]/[T/(nA− 1)], where T is the recording interval (min) that begins and ends with phase III propagating through the recording segment, n0 is the number of phases III at the oral side, and nA is the number of phases III propagating through the segment to the aboral side during T min fronts.

After completing the experimental protocol, the surgically modified rats were killed by isoflurane overdose and exsanguination. Postmortem examinations performed at the conclusion of the experiment revealed no signs of inflammation around the implanted electrodes and transmitters.

Tissue sampling

Prior to tissue sampling, food but not water was removed for 12 h from randomly chosen animals following 7 or 21 days of experimental treatment. The animals were then killed by isoflurane overdose and exsanguination. At death, the stomach, two specimens of small intestine i.e. jejunal sample (10–20 cm from the pylorus) and ileal sample (10 cm segment ending 2 cm proximal to caecum), caecum and colon were removed for isolation and enumeration of bacteria.

Isolation and enumeration of bacteria

GI tract samples (tissues plus contents) were weighed, and homogenized on ice with a Janke-Kunkel (Ascher, Netherlands) Ultra-Turrax T25 homogenizer at 20 000 r.p.m. in MRD (Oxoid) to provide a 100 g l−1 suspension, and serially diluted to 10−8 with the same diluent. Dilutions of the samples were then inoculated in duplicate onto selective and non-selective agars: Columbia agar (Oxoid) containing 5% (v/v) defibrinated horse blood (for total anaerobic bacteria); Rogosa agar (Oxoid) containing glacial acetic acid (1.32 ml l−1; for lactobacilli); and MacConkey agar no. 3 (for enterobacteria). Plates for anaerobic incubation had been stored in an anaerobic environment for 48 h. Two selective media were used for enumeration of bifidobacteria: Wilkins Chalgren agar (Oxoid) containing acetic acid (1 ml l−1) and Mupirocin (100 mg l−1; Oxoid), as described by Rada et al. (1999), and BSM (Leuschner et al. 2003). Inoculated agar plates were incubated in an anaerobic cabinet (Don Whitey Scientific, Shipley, UK) at 37°C (Columbia agar and Rogosa agar plates for 48 h; both bifidobacteria selective media plates for 72 h), except for MacConkey agar plates, which were incubated aerobically (at 37°C for 18 h).

The number of total anaerobic bacteria, lactobacilli, enterobacteria and bifidobacteria in the random samples of the control and experimental diet, taken on a weekly basis, was evaluated by dissolving 1 g of the diet in 100 ml of MRD (Oxoid). Serial dilutions were prepared using the same diluent and plating them in duplicate onto selective and non-selective agars as described above.

Statistics

Statistical analyses were performed using the SPSS statistics package (v.11.0.1 for Windows; SPSS, Woking, UK). Diet group differences and time effects on bacterial counts were evaluated using two-factor independent measures analysis of variance (ANOVA), with the diet and the duration of feeding the diet as the main factors. A significant interaction between diet and time indicated that the effects of the diet and time were interdependent. Where appropriate, Tukey's post hoc test was used to compare the diet effect for each of the time points studied. The temporal and spatial parameters of the MMC were analysed separately for each electrode using the following model:

display math

where Yijkl is the dependent variable, µ is the overall mean, αi is the effect of diet (non-supplemented or supplemented), βj is the effect of time on each diet (7 or 21 days), γk is the effect of recording (first or second), αβγijk is the interaction between diet, time on diet and recording, αβij the interaction between diet and time on diet, αγjk is the interaction between diet and recording, Uk is the random effect of animal (k = 1, . . . 6), and ɛijkl represents the unexplained random error, where l represents the repetitions on each animal. Diet, time on diet, recoding and their interactions were not significant (P > 0.05) and thus not included in the final statistical model. Therefore, the final statistical model included the effect of diet (non-supplemented or supplemented) and random animal effect (in order to account for repeated measurements on the same animal). Because the preliminary analysis revealed no statistically significant differences in any of the parameters between the two dietary supplements groups, these data were pooled for comparison with the unsupplemented data.

The level of statistical significance was assigned as P < 0.05 in all performed analyses. Data are expressed as means ±s.e.m.

Results

Animals

All rats appeared clinically healthy throughout the experiment. The mean food intake was 4.0 ± 0.1 g (100 g live body weight)−1, and there were no differences in food consumption between and within dietary groups.

Microbiological studies

Figure 3 shows the counts of total anaerobe, lactobacilli, bifidobacteria and enterobacteria in the stomach, jejunum and ileum. Dietary supplementation significantly increased the number of bifidobacteria in the stomach (Fig. 3A) and the small intestine (Fig. 3B and C) after 7 days of treatment. Furthermore, the number of bifidobacteria in the stomach increased with the duration of the treatment, being significantly higher after 21 than after 7 days of dietary supplementation. In both jejunum (Fig. 3B) and ileum (Fig. 3C) the number of enterobacteria decreased following 21 days of dietary supplementation with synbiotics. Supplementation of the diet with synbiotics did not have any significant effect on the number of total anaerobes, nor lactobacilli in the stomach (Fig. 3A), or in small intestine (Fig. 3B and C).

Figure 3.

Bacterial counts from stomach, jejunal and ileal tissue and content of rats fed control and experimental diet for 7 or 21 days
Total heights of filled and open bars represent mean values ±s.e.m. for rats fed control and experimental diet, respectively. t, d and t × d are P values for the effect of time, diet, or the interaction between time and diet, respectively. Values marked with an asterisk are significantly different from the control diet (**P < 0.01).

Figure 4 shows the counts of total anaerobe, lactobacilli, bifidobacteria and enterobacteria in the caecum and colon. In both caecum and colon, the number of bifidobacteria increased after 7 days, while the number of total anaerobes and lactobacilli increased after 21 days of the dietary supplementation with synbiotics. In both caecum and colon the number of enterobacteria decreased after 21 but not 7 days of the treatment.

Figure 4.

Bacterial count of caecum and colon tissue and content of rats fed control and experimental diet for 7 or 21 days
Total heights of filled and open bars represent mean values ±s.e.m. for rats fed control and experimental diet, respectively. t, d, and t × d are P values for the effect of time, diet, or the interaction between time and diet, respectively. Values marked with an asterisk are significantly different from the control diet (*P < 0.05; **P < 0.01; and ***P < 0.001).

Interdigestive myoelectric activity of the small intestine

Table 1 shows the temporal characteristics of the intestinal myoelectric activity in conscious, freely moving rats. In all rats, regardless of the dietary treatment, duodenojejunal myoelectric activity exhibited triple-phase MMC. The results in Table 1 demonstrate that dietary supplementation with synbiotics significantly shortened the duration of MMC cycles in both parts of the jejunum, i.e. 14 and 28 cm distal to pylorus. In both parts of the jejunum, the supplementation of the diet significantly decreased the duration of phase II of the MMC, while it did not change the duration of the phase I and phase III of the MMC. The durations of the duodenal MMC cycles and phases were not affected by the experimental treatment.

Table 1.  Effect of diet (C, non-supplemented; E, supplemented) on the duration of phases and cycles of the migrating myoelectric complex (MMC) at three levels of small intestine, 2 (D), 14 (J1) and 28 cm (J2) distal to the pylorus in rats
Duration (min)DJ1J2
Diet CDiet EDiet CDiet EDiet CDiet E
  1. Data are presented as means ±s.e.m.* Significantly different at P < 0.05 at respective intestine level.

Phase III 3.0 ± 0.1 3.1 ± 0.1 4.0 ± 0.13.8 ± 0.1 4.5 ± 0.14.3 ± 0.1
Phase I12.7 ± 1.211.2 ± 1.4 9.3 ± 0.98.7 ± 1.0 7.7 ± 0.67.3 ± 0.7
Phase II 3.9 ± 0.3 3.5 ± 0.3 6.4 ± 0.4 5.3 ± 0.4* 6.2 ± 0.3 5.0 ± 0.3*
MMC cycle19.1 ± 1.017.3 ± 1.220.6 ± 1.018.4 ± 0.8*19.2 ± 1.116.9 ± 1.1*

Treatment with synbiotics significantly increased the occurrence of phase III of the MMC in all three levels of the small intestine (Table 2). Furthermore, the aboral propagation ratio (R) between the number of phases III in the oral and aboral parts of the recording segment was significantly (P < 0.001) increased from 0.71 ± 0.01 to 0.93 ± 0.01 by supplementation of the diet with synbiotics. The propagation velocity of phase III in the whole recording segment (2–28 cm distal to the pylorus) was also significantly (P < 0.05) increased by experimental treatment from 3.7 ± 0.2 to 4.4 ± 0.2 cm min−1.

Table 2.  Effect of diet on the occurrence of phase III of the MMC (the percentage of phase III at each respective electrode site in relation to all occurring phases III in the recording segment) at three levels of small intestine, 2 (D), 14 (J1) and 28 cm (J2) distal to the pylorus in rats
Intestine levelDiet CDiet E
  1. Data are presented as means ±s.e.m.*** Significantly different at P < 0.001 at respective intestine level.

D74.8 ± 2.494.7 ± 2.4***
J174.7 ± 1.395.9 ± 1.2***
J266.4 ± 3.190.1 ± 3.3***

The frequency of response potentials of the propagated phases III was increased at both levels of the jejunum (P < 0.01 and P < 0.05 for the proximal and distal jejunum, respectively), but not in the duodenum (Table 3).

Table 3.  Effect of diet on the frequency of response potentials (spikes min−1) of the propagated phases III of the MMC at three levels of small intestine, 2 (D), 14 (J1) and 28 cm (J2) distal to the pylorus in rats
Intestine levelDiet CDiet E
  1. Data are presented as means ±s.e.m. Values marked with asterisks are significantly different at or **P < 0.01 at respective intestine level.

D373 ± 10394 ± 12
J1246 ± 11  296 ± 12**
J2336 ± 13  363 ± 10**

Discussion

This study shows that supplementation of the diet with the pro- and prebiotics produced concomitant changes in the characteristic of intestinal myoelectric activity and composition of the bacterial community in normal elderly rats. Changes in the composition of the gastrointestinal bacterial community after consumption of pro- and prebiotics are well documented in both animal and human studies (Gibson et al. 1995; Campbell et al. 1997; Gibson, 1999; Kruse et al. 1999). The prebiotic used in this study was shown in vitro to stimulate the survival and growth of both probiotic microorganisms (Femia et al. 2002). For probiotics, it has been suggested that a mixture of probiotic strains may be more effective on the intestine than the individual strains (Ouwehand et al. 2002).

In the present study the EMG measurement of the myoelectric activity of gastrointestinal small muscles in vivo was used to evaluate the characteristics of small intestinal motility. This method was one of the first to be developed (Szurszewski, 1969; Sarna, 1989), and it is still frequently used to investigate small intestinal motility, hence it has been shown that the force, duration and propagation of muscle contractions are directly related to amplitude, frequency and propagation of spike potentials (Szurszewski, 1987). In the present study we implemented a radiotelemetry technique that allows the quantification of intestinal EMG in freely moving, unrestrained rats. It was previously shown that radiotelemetry helps to exclude distress related to handling and restraining rats (for reviews see Kramer et al. 2001; Kramer & Kinter, 2003). Therefore, it is suggested that the use of the radiotelemetry allows the exclusion of both acute and chronic stress from experiments, and that the changes in the characteristics of intestinal MMC observed in the present study in rats were evoked by the experimental treatments and not by stress artefacts.

The use of radiotelemetry allowed the recording of intestinal EMG over a longer period of time during each recording session. Bränström & Hellström (1996) showed that, in order to reduce the variability of the MMC characteristics, a minimum of four MMC cycles and at least five phases III of the MMC should be recorded, i.e. approximately a minimum of 1.5 h of recording under the particular experimental conditions. In the present study we were able to record at least 6 h of interdigestive intestinal EMG, i.e. an average of 19 MMC cycles from each session on each animal. The reduction of the MMC variability, together with the fact that animals were used as their own controls, allowed a reduction in the number of the animals required for the experimental procedure (Festing, 1992). This is in agreement with the previous research results on measurements of different physiological functions when using the radiotelemetry technique in rats in vivo (Fraser et al. 2001).

In the present study the addition of the synbiotics to the diet produced significant changes in the temporal and spatial characteristics of the myoelectric activity of the small intestine. The duration of the jejunal MMC cycles was decreased at the expense of the shortening of phase II of the MMC. This suggests faster luminal transport in jejunum, hence the rate of the intestinal luminal passage depends on the migration rate of the MMC (Laplace & Roman, 1979). This is supported by the other MMC parameters obtained in the present study, i.e. higher migration velocity of phase III as well as higher frequency of response potentials of phase III observed in animals fed the diet supplemented with synbiotics. The increase in these two parameters of phase III indicate that the intestinal contractions during phase III occurred with higher frequency and were stronger, hence the amplitude and propagation of intestinal contraction is directly related to the frequency and propagation of spike potentials (Szurszewski, 1987). In the present study the supplementation of the diet with synbiotics significantly increased the aboral propagation ratio of phase III of the MMC, i.e. the contractions triggered in the duodenum migrated distally over significantly longer distances in the small intestine when rats were fed diet supplemented with synbiotics. This is in agreement with the results of the study by Huseby et al. (1994) on GF Spraque–Dawley rats, in which the conventionalization of gut microflora resulted in an increase in the calculated propagation ration of phase III in the small intestine. Overall changes observed in phase III characteristics in the present study indicate that the addition of the synbiotics to the diet stimulated a more regular occurrence of intestinal contractions of higher amplitude. Lumen occlusive and regular contractions are more effective in propelling the residual food, debris, secretions and bacteria cells (Code & Marlett, 1975). Therefore, we suggest that the addition of the synbiotics to the diet improved the interdigestive intestinal motility function in the rats our study.

In the present study the supplementation of the diet with synbiotics produced changes in the intestinal myoelectric activity from the seventh day of the experimental treatment, corresponding to the time of the increase in number of bifidobacteria in all parts of GI tract of the rats fed diet supplemented with synbiotics (Figs 3 and 4). Furthermore, the increase in the number of bifidobacteria in rats fed E diet was most prominent (59%) when compared to other evaluated bacterial groups, e.g. 9 and 11% increases in the number of lactobacilli and total anaerobes, respectively. There are no published data available on the relationship between dietary-evoked changes in the composition of the commensal intestinal microflora and changes in the characteristics of intestinal motility. Husebye et al. (2001) have shown that Lactobacillus acidophillus A10 and Bifidobacterium bifidum B11 accelerated small intestinal motility and digesta transit of adult GF rats. It may be that both bacterial strains evoked a specific response in terms of intestinal motility. Results from previous studies on the relationship between microflora and the GI tract motility showed that bacterial fermentation products, i.e. short-chain fatty acids (SCFA) regulate gastric (Cuche et al. 2000), ileal (Kamath et al. 1988) and colonic motility (Yajima, 1985; Cherbut et al. 1998) via humoral factors and neural pathways. This may indicate that different profiles of SCFA resulting from changes in the microflora may be involved in the regulation of duodenojejunal motility observed in rats in the present study. This, however, requires further investigation. Following 21 days of experimental treatment of animals with synbiotics there was a decrease in the numbers of enterobacteria in small intestine. This decrease did not show a relationship with the changes observed in the intestinal motility characteristics over time. However, there was a time association with the increase in the number of lactobacilli and bifidobacteria in the GI tract of rats fed synbiotics. It has been shown in vitro and in vivo in GF animals that both bifidobacteria and lactobacilli possess antagonistic activity against opportunistic bacteria, e.g. E. coli, and potentially pathogenic enterobacteria (Itoh & Freter, 1989; Korshunov et al. 1999; Bevilacqua et al. 2003; Asahara et al. 2004; Pêna et al. 2005). Therefore, it is more likely that decreases in the number of enterobacteria were related to the increase in the number of bifidobacteria and lactobacilli rather than to motility changes observed in a gut of rats fed synbiotics in the present study.

In conclusion, employing the radiotelemetry technique permitted the accurate recording and analysis of intestinal EMG in freely moving, unrestrained rats. Furthermore, using the telemetry technique it was demonstrated that changes in the gastrointestinal microflora exhibited an intestinal motility response and, more importantly, that such changes can be initiated by the addition of synbiotics to the diet. Further work is necessary in order to identify the underlying mechanisms responsible for diet/bacterial-induced changes in gastrointestinal motility. These findings may be of some relevance to the treatment of gastrointestinal dysfunction, especially that related to ageing, and this model may provide a means of studying these diseases further.

Appendix

Acknowledgements

This work was funded by the European Commission (Marie Curie Individual Fellowship, contract no. CT-2001-51915). Technical support provided by Donna Lecky, Kathrine Hansen Høirup and all staff from the Animal Intensive Care Unit at DIAS is gratefully acknowledged.

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