APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2010, p. 5592–5600 0099-2240/10/$12.00 doi:10.1128/AEM.00524-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 16
Development and Validation of a Continuous In Vitro System Reproducing Some Biotic and Abiotic Factors of the Veal Calf Intestine䌤 Marie Ge´rard-Champod,1,2† Ste´phanie Blanquet-Diot,1†* Jean-Michel Cardot,1 David Bravo,2 and Monique Alric1 Clermont Universite´, Universite´ d’Auvergne, Centre de Recherche en Nutrition Humaine Auvergne, ERT 18, Conception Inge´nierie et De´veloppement de l’Aliment et du Me´dicament, BP 10448, F-63000 Clermont-Ferrand, France,1 and Pancosma SA, Voie-des-Traz 6, C.P. 143, CH-1218 Le Grand-Saconnex, Switzerland2 Received 26 February 2010/Accepted 18 June 2010
Following the January 2006 European ban of antibiotics used as growth promoters in the veal calf industry, new feed additives are needed in order to maintain animal health and growth performance. As an alternative to in vivo experiments in the testing of such additives, an in vitro system modeling the intestinal ecosystem of the veal calf was developed. Stabilization of the main cultured microbial groups and their metabolic activity were tracked in an in vitro continuous fermentor operated under anaerobiosis, at pH 6.5, and at a temperature of 38.5°C and supplied with one of three different nutritive media (M1, M2, or M3). These media mainly differed in their concentrations of simple and complex carbohydrates and in their lipid sources. In vitro microbial levels and fermentative metabolite concentrations were compared to in vivo data, and the biochemical composition of the nutritive media was compared to that of the veal calf intestinal content. All three nutritive media were able to stabilize anaerobic and facultative anaerobic microflora, lactate-utilizing bacteria, bifidobacteria, lactobacilli, enterococci, and Bacteroides fragilis group bacteria at levels close to in vivo values. The microbiota was metabolically active, with high concentrations of lactate, ammonia, and short-chain fatty acids found in the fermentative medium. Comparison with in vivo data indicated that M3 outperformed M1 and M2 in simulating the conditions encountered in the veal calf intestine. This in vitro system would be useful in the prescreening of new feed additives by studying their effect on the intestinal microbiota levels and fermentative metabolite production. ders, as previously observed in pigs and poultry in the Nordic countries (36), where AGP have been totally prohibited since the 1990s. Even though no scientific study has yet been done on calves, there have already been reports of higher death rates on experimental commercial farms subsequent to the withdrawal of AGP. The main digestive diseases leading to veal calf deaths are enteritis and enterotoxemia, which are mainly triggered by pathogenic strains of Escherichia coli and Clostridium perfringens (22, 30). Veal calf producers are looking for new feed additives to allay the consequences of the AGP ban. Alternative approaches include the use of prebiotics, probiotics, or plant extracts. Several studies have reported both consistent improvements in weight gain and feed conversion and a reduction of the incidence of diarrhea with the addition of such additives to the veal calf diet (1, 11, 14). One of the hypotheses used to explain these beneficial effects involves the modulation of the intestinal microbiota. In particular, oligosaccharides containing mannose or fructose are known to selectively increase the growth of beneficial intestinal bacteria, including lactobacilli and bifidobacteria (21). Timmerman et al. (33) showed that a calf-specific probiotic containing six Lactobacillus species reduced the fecal counts of E. coli. Green tea extracts also improved the intestinal microbial balance by maintaining high fecal levels of Bifidobacterium and Lactobacillus spp. and decreasing those of C. perfringens (16). As indicated above, it is important to assess the action of
European regulations introduced in January 2006 banned the use of antibiotics as growth promoters (AGP) at subtherapeutic levels in animal feed (regulation EC 1831/2003), particularly for veal calves. AGP generated significantly enhanced growth performance via complex processes. The mechanism of growth promotion is still speculative, but many studies suggest the involvement of the intestinal microbiota (7, 9). First of all, AGP did not promote the growth of germfree animals (6). Moreover, they strongly inhibited the bacterial catabolism of urea and amino acids and the fermentation of carbohydrates both in vitro and in vivo (10, 28, 35). AGP treatment thus provided the animal with higher nutrient availability and led to a decrease in the toxic metabolites produced by bacteria, like ammonia or amines, limiting the energy needed by the animal to detoxify the organism. Some authors also argue that another beneficial effect of AGP results from improved control of intestinal pathologies, such as necrotic enteritis in poultry (12). The January 2006 ban is thus expected to have an impact on veal calf health by leading to more frequent digestive disor* Corresponding author. Mailing address: Equipe de Recherche Technologique, Conception Inge´nierie et De´veloppement de l’Aliment et du Me´dicament (ERT CIDAM), Universite´ d’Auvergne, Faculte´ de Pharmacie, 28 Place Henri Dunant, 63001 Clermont-Ferrand, France. Phone: (33) 473 17 83 90. Fax: (33) 473 17 83 92. E-mail:
[email protected]. † These authors contributed equally to this work. 䌤 Published ahead of print on 25 June 2010. 5592
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newly developed feed additives on the veal calf intestinal microbiota. High interindividual variability makes it difficult and expensive to carry out in vivo studies. Alternatively, experiments can be conducted via in vitro systems modeling the intestinal environment of the animals, provided the model has been checked as pertinent. This approach should allow an economical and ethical way to prescreen feed additives by studying their effects on the intestinal microbiota cultured in the in vitro system and its metabolic activity. With this objective in mind, a necessary requirement is knowledge of the veal calf intestinal ecosystem. Thus, the bacterial and biochemical composition of the jejunoileal chyme of calves was previously characterized (13). The aims of the present study were (i) to set up an in vitro system where the main cultured microbial groups identified in the veal calf intestinal chyme are reproducibly stabilized and metabolically active and (ii) to validate our model by comparing the in vitro and in vivo levels of selected biotic and abiotic variables. MATERIALS AND METHODS Animals. Twenty-seven calves of the Prim’Holstein breed (age, 22.3 ⫾ 1.5 weeks; average weight, 122 ⫾ 10 kg) were obtained from four different veal farmers running under controlled rearing conditions. The calves were fed a standard diet (13). They received no antibiotic therapy for at least the last four weeks running up to the experiments. The animal housing, husbandry, and slaughtering conditions were compliant with European Union standards (34). The intestinal contents were collected on the slaughtering line from animals whose meat was intended for human consumption. The animals were slaughtered 6 h after their last meal. Preparation of inocula for in vitro fermentation. Jejunoileal samples were taken from the intestines of three animals within 5 min after slaughter (13). The contents were immediately brought to the lab in CO2-filled bottles and treated in an anaerobic chamber. Conditions inside the chamber were maintained using commercial premixed gas containing 90% N2, 5% CO2, and 5% H2 (AirProducts, Paris, France). Anaerobiosis was constantly monitored on an oxygen analyzer (Coy Laboratory Products, Grass Lake, MI), and O2 levels were maintained at 10 ppm or less using palladium catalysts. The contents were filtered through gauze to remove the largest particles, such as hair. A homogenized pool of the three samples (400 ml each) was used as the inoculum for the in vitro system. A 5-ml inoculum sample was taken to perform microbial counts at the initial time of fermentation (day 0), and 17 ml was frozen (⫺20°C) for further metabolite analyses. In vitro fermentation conditions. The experiments were carried out in a continuous culture system (Bioflo 3000; New Brunswick, Paris, France). The system comprised one glass vessel with an operating volume kept at a constant 1,200 ml by a level probe controlling the outflow of the fermentative content. A sterile nutritive medium made fresh daily was introduced continuously at a rate of 15 ml/h (retention time, 80 h). Three different media (M1, M2, and M3) were supplied to the fermentor (see Table 1 for composition). The fermentative medium was permanently stirred by a screw at a rate of 255 rpm, the pH was kept at a constant 6.5 by adding 3 M NaOH, and the temperature was maintained at 38.5°C. The atmosphere was filtered (0.2 m) continuously. During the preparation of the inoculum, the in vitro system was flushed with N2 to create an anaerobic atmosphere. The inoculum was transferred from the anaerobic chamber to the fermentor in a hermetically sealed bottle, and fermentation started immediately. N2 flushing was stopped, and the anaerobic conditions inside the vessel were maintained by microbial activity. The anaerobic state of the vessel was controlled daily by measuring O2 levels via gas chromatography (HP 6890 series columns Molecular Sieve 5A and Porapak Q; Agilent Technologies, Santa Clara, CA). Every day, 22 ml of fermentative medium was collected. Five milliliters was used for microbial counts, and 17 ml was frozen (⫺20°C) for further metabolite analyses. Each experimental condition, i.e., with each different nutritive medium, was tested three times (for a total of nine runs), and the experiments lasted 7 days. Biochemical composition of the nutritive media. The biochemical composition of the three nutritive media was analyzed according to Ge´rard-Champod et al. (13). Briefly, humidity was determined by the Karl Fisher method and used to
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TABLE 1. Composition of the three nutritive media provided to the in vitro system Concna in: Component M1
Carbohydrates Glucose Galactose Lactose Apple pectin Birch wood xylan Corn starch Larch wood arabinogalactan Microcrystalline cellulose Arabic gum Guar gum Potato starch Porcine stomach mucin Proteins Primatone Bacto tryptone Casein Yeast extract L-Cysteine hydrochloride Lipids Bile salts Tween 80 Tallow Copra oil (100% hydrogenated) Palm oil (100% hydrogenated) Soya oil (45% hydrogenated) Sunflower oil Soya lecithin
b
8 6 6 6 8 6 6 44 15 25 25.0 0.4 0.4 20
M2
3 5 3 8 6 6 6
3 5 3 6
6 6 38 30
33 45
20 12.5 12.5 5 0.4
17 12.5 12.5 5 0.4
0.4
0.4
5 0.9 6.6 1.3 4.4 1.8
5 0.9 6.6 1.3 4.4 1.8 1.34 2.8 0.177 0.005 0.02 3.0 4.00 1.0 E-6 2.5 E-7 5.0 E-6 2.5 E-6 2.5 E-6 2.5 E-6 5.0 E-7 0.75
Minerals and vitamins K2HPO4 䡠 3H2O NaCl KH2PO4 FeSO4 䡠 7H2O Hemin MgSO4 䡠 7H2O CaCl2 䡠 2H2O D-Biotin Vitamin B12 D-Pantothenic acid Nicotinamide 4-Aminobenzoic acid Thiamine Menadione
0.005 0.02 0.5 0.45 1.0 E-6 2.5 E-7 5.0 E-6 2.5 E-6 2.5 E-6 2.5 E-6 5.0 E-7
1.34 2.8 0.177 0.005 0.02 3.0 4.00 1.0 E-6 2.5 E-7 5.0 E-6 2.5 E-6 2.5 E-6 2.5 E-6 5.0 E-7
Antifoam A
0.75
0.75
2.50 4.5
M3
a
All values are in grams per liter, except those for antifoam A, which are in milliliters per liter. b Data adapted from Macfarlane et al. (20).
calculate the amount of dry matter. Total nitrogen was measured using the Kjeldahl method (ISO5983), and total protein was calculated by multiplying total nitrogen by the Jones factor (5.41). Amino acid profiles were determined on a Biochrom20 amino acid analyzer (Biochrom, Cambridge, United Kingdom). Lactose, glucose, and galactose were determined with enzymatic kits (R-Biopharm, Darmstadt, Germany). Glucose and galactose were measured in both raw and hydrolyzed samples. Fat content was measured after Soxhlet extraction with petroleum ether (3). Ash content was quantified in dried samples (550°C). Microbial counts. Anaerobic microflora, Bacteroides fragilis group bacteria, lactate-utilizing bacteria, lactobacilli, enterococci, bifidobacteria, E. coli, and C. perfringens were enumerated on selective solid culture media (13). Briefly, serial 10-fold dilutions were done in anaerobic, aerobic, or tryptone-glucose-yeast dilution medium. For anaerobic microflora (culture medium, brain heart infu-
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FIG. 1. Biochemical composition of the three nutritive media used in this study (n ⫽ 1) and that of the intestinal content of veal calves (n ⫽ 12). Results are expressed as average percentages ⫾ the standard error of the mean of dry matter. Significant differences between nutritive media and intestinal contents are indicated (ⴱ, P ⬍ 0.05).
sion agar), B. fragilis group bacteria (Bacteroides mineral salt agar), and C. perfringens (Viande Levure medium), 100 l of each serial dilution was inoculated into the anaerobic chamber using the spread plate method. For lactobacilli, the dilutions were maintained under CO2 flow and 1 ml of each serial dilution was inoculated onto Rogosa medium using the pour plate method. The roll tube technique (18) was used to count lactate-utilizing bacteria (lactate medium). For enterococci (bile esculin azide agar medium) and bifidobacteria (Beerens medium), 100-l volumes of the dilutions were inoculated under aerobic conditions via the spread plate method. Finally, E. coli was inoculated onto tryptone bile X-glucuronide medium under serial aerobic dilutions using the pour plate method. For facultative anaerobic microflora and yeast, serial 10-fold dilutions were done in aerobic dilution medium, brain heart infusion agar (29), and Sabouraud (24) medium, respectively, which were used for counting. The culture media were inoculated with 100 l of each serial dilution under aerobic conditions using the spread plate method. All of the media were incubated at 37°C, except for yeast enumeration (30°C). Members of the microbiota were enumerated by visual counting, taking mediumspecific discrimination into account. Fermentative metabolite analyses. Thawed samples were brought to 80°C in 15 min in a water bath to stop enzymatic reactions and then centrifuged (9,000 ⫻ g, 20 min, 4°C) before analyzing the supernatants for L-lactate and ammonia concentrations using enzymatic kits (R-Biopharm, Darmstadt, Germany). Shortchain fatty acid (SCFA) analyses were run via gas chromatography (HP 6890 series HP19091N-133 column in polyethylene glycol [30 m by 250 m by 0.25 m]; split ratio of 3:1; 140°C for 7 min, followed by 240°C for 4 min; Agilent Technologies, Santa Clara, CA) according to Jouany (17). Acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids were analyzed in thawed samples after deproteinization with mercury chloride I and centrifugation (15,000 ⫻ g, 15 min, 7°C). The internal standard used was 4-methylvaleric acid. Statistical analysis plan. (i) Descriptive analyses. The main study objective was to obtain experimental conditions allowing the stabilization of most of the microbial populations enumerated at levels close to those observed in vivo and with the best intertrial reproducibility. Steady state of a nutritive medium (M), a trial (T), and a microbial population (P) was defined as a succession of at least three time points with a coefficient of variation (CV) of ⬍10% and a global range lower than 2 logs. The beginning of each steady state was characterized by the TiMPT value (initial time of the steady state for each nutritive medium, bacterial population, and trial). TiMP (maximum TiMPT among the three trials) was the
time point from which the microbial population P was considered stable in medium M. TiM (maximum TiMP among all microbial populations) was the time point from which the overall selected populations were considered stable in medium M. (ii) Statistical analyses. Analyses of variance (ANOVA) were carried out using the general linear model procedure bundled with the SAS software (SAS Institute, Cary, NC) to study the variability of the microbial populations’ steadystate levels (i) between trials for a nutritive medium and (ii) between nutritive media (M1, M2, and M3) for the three trials. Fermentative metabolite concentrations were compared to the initial values and between the nutritive media using the unpaired Student t test. One-way ANOVA was used to test for significant differences between groups. Using the same method, in vitro microbial levels and fermentative metabolite concentrations were compared to in vivo data and the biochemical profiles of the nutritive media were compared against the jejunoileal contents of veal calves. A probability level of P ⬍ 0.05 was considered statistically significant.
RESULTS Biochemical composition of the nutritive media. The biochemical analyses of the three nutritive media (n ⫽ 1) are given in Fig. 1. Similar concentrations of total nitrogen (5%), protein (30%), and ash (8%) were observed in the three nutritive media, but M1 contained less fat than did M2 and M3 (3.0% versus 11.4 and 12.5%, respectively). Lactose, glucose, and galactose were found in lower concentrations in M1 (between 0.1 and 0.2% of the dry matter) than in M2 or M3 (between 1.4 and 3.1%). In the three media, acid hydrolysis led to an increase in glucose and galactose percentages (to approximately 25 and 10%, respectively). The amino acid profiles were similar for M1, M2, and M3 (data not shown) and showed a main peak consisting of two amino acids, glutamic acid and glutamine, as our analytical method did not allow their separation. Fatty acid profiles were highly different between the nutritive media (data not shown). M1 was mainly composed of
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FIG. 2. Concentrations of the selected microbial groups during fermentations with M1 (in black), M2 (in gray), or M3 (in white). Results are expressed in log10 CFU/ml of fermentative medium ⫾ the standard error of the mean (n ⫽ 3). TiMPs are represented by arrows.
oleic acid, whereas M2 and M3 were similar to each other, with four major fatty acids (palmitic, stearic, oleic, and linoleic acids). In vitro levels of selected microbial groups. The average concentrations (n ⫽ 3) of the enumerated microbial groups are presented in Fig. 2. All three nutritive media followed the same
trends. Anaerobic microflora and lactate-utilizing bacteria stabilized between 8 and 10 logs; facultative anaerobic microflora stabilized between 7 and 9 logs; lactobacilli, bifidobacteria, and enterococci stabilized between 6 and 8 logs; and B. fragilis group bacteria stabilized between 5 and 7 logs. All of these bacterial groups stabilized around their initial levels (day 0). E.
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TABLE 2. Effect of nutritive medium on the stabilization of the selected bacterial groups during in vitro fermentations M1 (n ⫽ 3)
M2 (n ⫽ 3)
M3 (n ⫽ 3)
Bacterial group
No. of steady statesa/total
TiMPb (days)
Intertrial CVc (%)
No. of steady states/total
TiMP (days)
Intertrial CV (%)
No. of steady states/total
TiMP (days)
Intertrial CV (%)
Anaerobic microflora Facultative anaerobic microflora Lactate-utilizing bacteria Lactobacilli Bifidobacteria Enterococci B. fragilis group E. coli
3/3 1/1
0 0
9 NDd
3/3 1/3
0 0
7 ND
3/3 3/3
0 3
12 29
2/3
4
19
3/3
0
8
3/3
3
30
3/3 2/3 2/3 3/3 1/3
0 2 5 5 4
24 44 7 29 ND
2/3 1/3 2/3 1/3 2/3
0 2 3 1 4
16 ND 49 ND 40
2/3 2/3 2/3 2/3 2/3
4 3 4 4 5
14 16 40 25 1
Steady state is defined as a succession of at least three time points with a CV of ⬍10% and a global range lower than 2 logs. Initial time of the steady state for a bacterial population and a nutritive medium. CV calculated during steady state. d ND, not determined. a b c
coli stabilized between 4 and 6 logs, which was, on the contrary, far from the initial value (7.5 logs). C. perfringens and yeasts disappeared from the fermentative medium by day 2. These two groups were not statistically analyzed, as they stabilized close to the zero level. Table 2 presents the number of stabilized trials over the three repetitions for each bacterial population, giving the initial time of the steady state (TiMP) and the intertrial CV. TiMPs are also shown in Fig. 2. M1 and M3 were able to stabilize selected bacterial populations in at least two out of three trials, except for E. coli, which was stabilized only once with M1. M2 appeared to be the least efficient medium, as three of the enumerated populations were stabilized only once and only two populations (anaerobic microflora and lactate-utilizing bacteria) were stabilized in all of the trials. The time needed for all of the enumerated bacterial populations to reach a steady state (TiM [maximum TiMP]) was 5 days for M1 and M3 and 4 days for M2. Analysis of the intertrial CV during the steady states showed that the variability was lower for the anaerobic microflora (from 7% to 12%, depending on the nutritive medium) than for the other bacterial groups (from 1% to 49%). M1 led to the lowest CV for enterococci, M2 for the anaerobic microflora and lactate-utilizing bacteria, and M3 for all of the other populations. In vitro concentrations of fermentative metabolites. L-Lactate, ammonia, and SCFA concentrations were measured every day during the fermentations with M1, M2, and M3 (Fig. 3). L-Lactate concentrations decreased from day 0 to day 1 in all of the trials and then increased from day 4 to the end of the experiment with M2 and M3 only. With M1, L-lactate was undetectable after day 1. There was a significant difference in L-lactate concentrations between M1 and the other two nutritive media during the microbial steady state (P ⱕ 0.009). In all three trials, ammonia levels increased from day 0 (global average, 0.23 ⫾ 0.05 g/liter) to day 2 and remained stable thereafter with M1 and M2. During the microbial steady state, there were significantly (P ⱕ 0.016) higher concentrations of ammonia with M3 than with M1 or M2. Total SCFA amounts increased strongly in all three trials from day 0 (global average, 40 mM) to day 2 (266 mM). Then, with M3, the SCFA concentration stabilized at approximately 200 mM until the end of
the experiment, whereas with M1 and M2, the total SCFA decreased from day 4 to day 7. Comparison with in vivo data. Nitrogen, protein, and ash composition was not significantly different (P ⬎ 0.05) between the calf jejunoileal content and the three media (Fig. 1). How-
FIG. 3. Concentrations of the fermentation products during in vitro assays with M1 (black circles), M2 (gray circles), or M3 (white circles). Results are expressed in g/liter ⫾ the standard error of the mean (n ⫽ 3) of fermentative medium for L-lactate and ammonia and in mM ⫾ the standard error of the mean (n ⫽ 3) for total SCFA. TiMs are represented by arrows. Significant differences (P ⬍ 0.05) between M1 and the other nutritive media (ⴱ) or between M3 and the other nutritive media (†) are indicated.
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FIG. 4. Comparison of in vitro and in vivo levels of selected bacterial populations. In vitro microbial levels obtained with M1 (A), M2 (B), and M3 (C) during the steady states (in gray; n ⫽ number of points during the stabilized states) were compared to the in vivo database (in white; n ⫽ 12). The box plot represents the interquartile range. The solid line represents the average of the values, and the dotted line represents the median. Minima and maxima are represented by symbols (} and Œ, respectively). Significant differences between in vitro and in vivo data are indicated (ⴱ, P ⬍ 0.05).
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FIG. 5. Comparison of in vitro and in vivo levels of fermentative metabolites. Comparisons are made between metabolite concentrations found during the in vitro microbial steady states (in gray, n ⫽ number of points during the stabilized states) and those obtained in the veal calf (in white, n ⫽ 12). The box plot represents the interquartile range. The solid line represents the average of the values, and the dotted line represents the median. Minima and maxima are represented by symbols (} and Œ, respectively). Significant differences between in vitro and in vivo data are indicated (ⴱ, P ⬍ 0.05).
ever, fat, glucose, galactose, and lactose concentrations were lower in M1 (but not M2 or M3) than in vivo. Moreover, in contrast with M2 and M3, we noted differences between the fatty acid profile of M1 and that recorded in vivo (data not shown). In particular, the complexity found in the jejunoileal content was not recovered in M1, which was mainly composed of oleic acid. Figures 4 and 5 illustrate the comparative analysis of the microbial and metabolite concentrations obtained during stabilized states of in vitro fermentations and the concentrations measured in the veal calves. During fermentations with M1 (Fig. 4A), there were no statistically significant differences between the levels of microbial populations stabilized in the fermentor and the in vivo levels of all of the bacterial groups enumerated (P ⬎ 0.13), except for the anaerobic microflora, which was stabilized at a significantly higher concentration in vitro than in vivo (P ⬍ 0.001). The median was always located on the same side of the average in vivo and in vitro, showing a similar data distribution. With M2 (Fig. 4B), anaerobic microflora and lactate-utilizing bacteria were stabilized at significantly higher concentrations in vitro than in vivo (P ⱕ 0.001). For most of the bacterial groups, the medians were not on the same side of the average in vivo and in vitro, which indicates that the values were inversely distributed in vitro and in vivo. With M3 (Fig. 4C), no significant difference was observed between the in vitro steady states and the in vivo data for enterococci, bifidobacteria, and lactate-utilizing bacteria. Lactobacilli were stabilized at a higher value in the in vitro system than in vivo (P ⫽ 0.01), whereas the in vitro concentrations of B. fragilis group bacteria and E. coli were significantly lower than the in vivo values (P ⱕ 0.01). Variance analysis of anaerobic microflora indicated a significant difference between the two groups, but as the averages and medians differed by less
than 1 log and the interquartile ranges overlapped, the difference was not considered significant. Concerning fermentative metabolites (Fig. 5), the statistical analyses showed a marginal but nonsignificant difference (P ⱖ 0.06) between in vivo and in vitro levels of L-lactate in M2 and M3 due to a high CV (⬎88%). L-Lactate concentrations during the microbial steady states in M1 were null for all of the trials, leading to a lower level in vitro than in vivo (P ⫽ 0.02). Ammonia and total SCFA were stabilized at significantly higher concentrations in vitro than in the veal calves (P ⬍ 0.001) for all of the nutritive media. DISCUSSION The aim of this study was to develop an in vitro model of the veal calf small intestine by stabilizing the main cultured bacterial groups present in this ecosystem at levels close to those found in vivo and keeping them metabolically active. The physicochemical parameters of the in vitro system were chosen according to in vivo data (13): the pH was controlled at 6.5, and the temperature was set at 38.5°C. According to preliminary results, the residence time was set at 80 h to avoid rapid flushing of some bacterial species. Three different nutritive media were tested. M1 is the medium classically used in in vitro models of the human gut (20). M2 and M3 were modified from M1 on the basis of biochemical analyses of the jejunoileal contents of veal calves (13). Briefly, the simple lipid source used in M1 (Tween 80) was replaced with a complex one in M2 and M3 to better represent the complexity found in vivo, and M2 and M3 were completed with simple sugars found in the intestinal contents of calves. It is assumed that glucose and galactose (the amounts of which increased after acidic hydrolysis) are derived from mucin, which is the main endogenous
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glycoprotein found in vivo (26). Compared to M2, M3 medium was devoid of fibers, as they are present in the veal calf diet in very small amounts. Biochemical analyses of the three nutritive media confirmed that M2 and M3 have a composition clearly closer to that found in vivo than does that of M1 and therefore appeared to be the best suited to mimic nutrient supplies in the veal calf small intestine. The facultative anaerobic microflora, anaerobic microflora, and taxonomic microbial groups enumerated in the in vitro system were chosen as marker populations (23). E. coli and C. perfringens bacteria were counted because they are involved in the main digestive diseases affecting calves (22, 30). Our fermentative conditions stabilized the selected microbial groups at a level close to the initial value, with the exception of E. coli. The main cultured genera found in the veal calf were therefore preserved in the in vitro system. In contrast, C. perfringens and yeast cells were rapidly cleared from the fermentor, probably due to their low concentrations in the inoculum compared to the other microbial groups enumerated (103 to 105 versus 107 to 108). A regular input of these species into the in vitro system could be a solution that would allow them to be maintained at the initial concentrations. Another way to avoid flushing of microbial species would be to improve the model by fixing the intestinal microbiota in gel beads, as already described by Cinquin et al. (5). Statistical analyses were performed in order to select one nutritive medium from among the three tested. Under our stringent analysis conditions (steady state defined by a ⬍10% CV), none of the media was able to stabilize all of the selected microbial populations in all of the trials. Nevertheless, based upon the results obtained, it was obvious that M1 and M3 outperformed M2. Considering the intertrial CV calculated during the steady states, M3 led to the most reproducible results for the majority (five out of eight) of the bacterial groups enumerated. The results obtained in vitro during the stabilized states were afterwards compared to those obtained previously in the animals (13). M1 was the medium that allowed the stabilization of selected microbial populations at the levels closest to in vivo values. Moreover, for all of the media, the ranges of concentrations observed during the in vitro steady states were equal to or even lower than those found in the veal calf. This indicates that modeling led to a decrease in the interindividual variability observed in vivo. Another important step in the setup of the in vitro model was to ensure that the microbiota stabilized in the fermentor was metabolically active. All of the experiments yielded fermentation products such as lactate, ammonia, and SCFA but with different trends. Lactate is an intermediate of the bacterial metabolism of carbohydrates used in the production of fatty acids (31). The difference in lactate concentrations between M1 and the other two media may be due to a higher availability of simple sugars in these two media. Consequently, it can be supposed that L-lactate was produced rather than consumed (by lactate-utilizing bacteria) in M2 and M3 and, inversely, consumed rather than produced in M1. Ammonia accumulated during fermentation at significantly higher levels than at day 0. High concentrations of this metabolite could become toxic for the microbiota (27). Nevertheless, the amounts reached in the in vitro system have been shown to be nontoxic for E. coli (27), C. perfringens (19), and yeasts (15), which were the only pop-
ulations that decreased or disappeared from the system during fermentation. Compared to the other two nutritive media, M3 allowed stabilization of the total SCFA concentrations. Several authors (2, 8, 25) have used this parameter to determine the steady state of their in vitro systems, implying a great advantage for M3 in this study. There was no significant difference between in vitro and in vivo data for lactate concentrations (except for M1), due to high variability in both cases. Inversely, ammonia and total SCFA were significantly higher in vitro than in vivo. This accumulation may be explained by the fact that our model does not reproduce the absorption of fermentation products such as that which occurred in vivo (32). Upcoming system optimizations should include the addition of a dialysis system to ensure the absorption of excess fermentation products, as has already been done in the dynamic in vitro system TIM-2 from TNO (4). This report describes the setup of the first in vitro system modeling selected biotic and abiotic parameters of the veal calf intestine. Our in vitro conditions led to the stabilization of a metabolically active and complex microbiota at levels close to in vivo values. Comparison between in vitro and in vivo data indicated that M3 medium gave better results than M1 and M2. This model should be used to prescreen new feed additives by studying their effects on the intestinal ecosystem of veal calves, particularly on the levels of the main cultured bacterial groups and on the concentrations of the main fermentative metabolites. ACKNOWLEDGMENTS This work was supported by a CIFRE grant from Pancosma SA to Marie Ge´rard-Champod. We are grateful to Sophie Gonzalez-Bafoil, Antoine Bernardin, and Ce´dric Coustet for technical assistance. REFERENCES 1. Abe, F., N. Ishibashi, and S. Shimamura. 1995. Effect of administration of bifidobacteria and lactic acid bacteria to newborn calves and piglets. J. Dairy Sci. 78:2838–2846. 2. Allison, C., C. McFarlane, and G. T. MacFarlane. 1989. Studies on mixed populations of human intestinal bacteria grown in single-stage and multistage continuous culture systems. Appl. Environ. Microbiol. 55:672–678. 3. Bedo ¨, S., P. Hagony, and J. Harczi. 1976. Studies of the methods used for the determination of the digestibility of crude fats in calves. Arch. Tierernahr. 26:179–193. 4. Blanquet, S., J. P. Meunier, M. Minekus, S. Marol-Bonnin, and M. Alric. 2003. Recombinant Saccharomyces cerevisiae expressing P450 in artificial digestive systems: a model for biodetoxication in the human digestive environment. Appl. Environ. Microbiol. 69:2884–2892. 5. Cinquin, C., G. Le Blay, I. Fliss, and C. Lacroix. 2004. Immobilization of infant fecal microbiota and utilization in an in vitro colonic fermentation model. Microb. Ecol. 48:128–138. 6. Coates, M. E., R. Fuller, G. F. Harrison, M. Lev, and S. F. Suffolk. 1963. A comparison of the growth of chicks in the Gustafsson germ-free apparatus and in a conventional environment, with and without dietary supplements of penicillin. Br. J. Nutr. 17:141–150. 7. Corpet, D. E. 2000. Me´canismes de la promotion de croissance des animaux par les additifs alimentaires antibiotiques. Rev. Med. Vet. 151:99–104. 8. De Boever, P., B. Deplancke, and W. Verstraete. 2000. Fermentation by gut microbiota cultured in a simulator of the human intestinal microbial ecosystem is improved by supplementing a soygerm powder. J. Nutr. 130:2599– 2606. 9. Dibner, J. J., and J. D. Richards. 2005. Antibiotic growth promoters in agriculture: history and mode of action. Poult. Sci. 84:634–643. 10. Dierick, N. A., I. J. Vervaeke, J. A. Decuypere, and H. K. Henderickx. 1986. Influence of the gut flora and some growth-promoting feed additives on nitrogen metabolism in pigs. II. Studies in vivo. Livest. Prod. Sci. 14:177–193. 11. Donovan, D. C., S. T. Franklin, C. C. Chase, and A. R. Hippen. 2002. Growth and health of Holstein calves fed milk replacers supplemented with antibiotics or Enteroguard. J. Dairy Sci. 85:947–950. 12. Elwinger, K., E. Berndtson, B. Engstrom, O. Fossum, and L. Waldenstedt.
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