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Nutritional and sanitary statuses alter postweaning development of caecal microbial activity in the rabbit
Comparative Biochemistry and Physiology, Part A 139 (2004) 293 – 300 www.elsevier.com/locate/cbpa
Nutritional and sanitary statuses alter postweaning development of caecal microbial activity in the rabbit Nadia Bennegadi-Laurenta, Thierry Gidenneb,*, Dominique Licoisc a
ENSAT, De´partement des Sciences Animales, BP 107, 31326 Auzeville–Tolosane, Castanet-Tolosan Cedex, France b Station de Recherches Cunicoles, INRA Toulouse, Ch. de Borderouge, BP 27, 31326 Castanet-Tolosan, France c Bioagresseurs Sante´ Environnement, e´quipe pathologie cunicole, INRA Tours, 37380 Nouzilly, France Received 9 June 2004; received in revised form 3 September 2004; accepted 14 September 2004
Abstract The postweaning development of caecal microbial activity was studied in the rabbit according to the sanitary status (conventional bCQ vs. specified pathogen-free bSPFQ) and the nutritional status (standard-fibre bSFQ vs. deficient-fibre bDFQ diet). The two diets were distributed ad libitum from weaning (28 days) to 70 days of age, respectively, to 80 C and 72 SPF rabbits. From 28 to 42 days, the volatile fatty acids concentration in the caecum (tVFA) of C rabbits was 50 mM/L and increased by 46% between 42 and 56 days, without interactions with the diet effect. In parallel, the bacterial fibrolytic activity decreased for xylanase and CMCase (32% and 60%, respectively, Pb0.05), while pectinase activity decreased more regularly from 28 to 70 days (28%, Pb0.05). At weaning, tVFA was similar among C or SPF rabbits, while at 70 days, it decreased by 23% for SPF and increased in C group (+31%). Cellulasic and hemicellulasic activity of bacteria were two to three times lower, respectively, in SPF rabbits compared to conventional ones. No interaction was detected between sanitary and nutritional status at 70 days of age for the caecal fermentative activity. With the FD diet, tVFA decreased by 10%, while butyrate proportion increased by 37% (at 70 days), whatever the sanitary status. In 70-day-old rabbits (C or SPF group), pectinasic activity was reduced by 30% when rabbits were fed the FD compared to the SF one. D 2004 Elsevier Inc. All rights reserved. Keywords: Sanitary status; Caecal fermentation; Fibrolytic activity; Fibre deficiency; Young rabbit
1. Introduction The caecum of the rabbit is the main site of fermentative activity related to the presence of an abundant microbial flora (Gouet and Fonty, 1979), which develops significantly from 3 weeks of age, as the feed intake of the young rabbit shifts from milk to dry feed (Padilha et al., 1995). The flora development is however affected by the sanitary status, as shown by Bennegadi et al. (2003) who compared conventionally bred rabbits to those bred in a protected environment characterised by an absence of the main pathogenic * Corresponding author. Tel.: +33 5 61 28 51 03; fax: +33 5 61 28 53 19. E-mail address: [email protected] (T. Gidenne). 1095-6433/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2004.09.022
agents (specified pathogen-free bSPFQ; Coudert et al., 1988). Padilha et al. (1995) reported low caecal fermentation at weaning for SPF rabbits compared to previous studies on conventional rabbits. Bennegadi et al. (2001) reported that a dietary fibre deficiency favoured the occurrence of digestive troubles in the growing rabbit, but with a lower incidence for SPF animals compared to conventional ones. A role of caecal microbial activity has been hypothesised but not studied. Therefore, this study aimed to analyse the interactions between sanitary [SPF vs. conventional (C)] and nutritional statuses (diet with a standard-fibre vs. a fibre-deficient level) of the young rabbit on development of the caecal microbial activity assessed in three ways: fermentation end-products levels, bacterial fibrolytic activity, and fibre digestion.
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2. Material and methods 2.1. Animals, diets, and experimental design Two groups of hybrid rabbits (Oryctolagus cuniculus, strain INRA A1067) were bred according to two sanitary status, either in a conventional experimental unit (group C, n=80 rabbits) or in a highly protected environment (group SPF, n=72 rabbits). SPF environment is obtained according to the method of Coudert et al. (1988), that includes several criteria, such as ventilation with pressured air filtered at 10 Am, specific staff, and a regular confirmation that animals are free of coccidia, oxyuris, Pasteurella, Clostridium spiroforme and enteropathogenic Escherichia coli. Conventional environment is close to a standard breeding unit, with a closed ventilated and heated breeding room, but without microbiological control. Conventional animals were protected from coccidia as the feeds contain a coccidiostat (Table 1), and they had a low E. coli flora as shown in a previous microbiological study on the same rabbits (Bennegadi et al., 2003). From weaning (day 28) until 70 days old, rabbits were placed in separated rooms (conventional or SPF) and housed in individual wired cages (dimensions, 263365 cm, lhb) at room temperature (18F2 8C) with feedboxes and water pipes, a 12:12 light/dark cycle (lights on 07:00 h), and a minimum of 0.8 m3 air changes per hour. Before weaning, litters and does were fed a commercial diet. At weaning, C and SPF groups were allotted into two subgroups, according to the litter origin and weaning
Table 1 Ingredients and chemical composition of the experimental diets Diets Ingredients (%) Dehydrated alfalfa meal Dehydrated beet pulp Wheat bran Wheat Soya bean meal Wheat straw DL methionine Minerals Oligo element and vitaminsa
Control (SF) 30.0 20.0 20.0 12.4 10.0 6.0 0.2 0.9 0.5
Fibre deficient (FD) 9.0 7.5 5.0 54.3 19.0 2.0 0.2 2.5 0.5
weight, and they were fed ad libitum either a diet having a standard-fibre concentration (diet SF, 30 and 26 rabbits, respectively, for C and SPF groups) or a fibre-deficient diet (diet FD, 50 and 46 rabbits, respectively, for C and SPF groups). The two experimental diets were prepared in pelleted form and formulated to ensure no changes in fibre quality, but only in fibre quantity. For instance, the lignocellulosic content of the diet SF (189 g/kg) corresponded to current recommendations (Gidenne, 2003), while that of the FD diet was only 88 g/kg. The origin and proportions of fibre fractions were similar among diets, as well as the ratio of digestible protein/digestible energy (Table 1). Feeds and water were available ad libitum. 2.2. Measurements of digestive efficiency and microbial activity The digestive efficiency was measured on healthy conventional and SPF rabbits from 42 to 46 days with the two experimental diets (12 rabbits/diet/sanitary status) according to the European reference procedure (Perez et al., 1995). The caecal microbial activity was assessed through the analysis of fermentation end-products and of bacterial fibrolytic activity. Caecal contents were obtained after a lethal injection of anaesthetic solution (sodium thiopental) in the marginal ear vein at the end of the caecotrophy period (about 11:00 h), at 28, 42, 56, and 70 days in conventional rabbits (n=12/diet at each age), and at 28 and 70 days in SPF rabbits (n=12/diet at each age). The pH of the caecal digesta was taken immediately after laparotomy, with a glass electrode pH meter (pH 95, WTW, Weilherm, RFA). Portions of caecal digesta sample (1–3 g fresh matter) were collected in three different tubes containing, respectively, 10 mL of 0.025M of an anaerobic buffer MES-DTT, 2 ml of H3PO4 (2%, v/v), and 3 ml of H2SO4 (2%, v/v), which were storage solution for further analysis of bacterial fibrolytic activity bBFAQ (Gidenne et al., 2002), volatile fatty acids bVFAQ (Bellier, 1994), and NH3–N (Verdow et al., 1977), respectively. The portion of the caecal content for BFA analysis was stored at 80 8C and at 20 8C for VFA and NH3–N until analysis. The duration of the collection procedure did not exceed 15 min. 2.3. Biochemical analysis
Chemical analysis (g kg 1 air dry basis) Dry matter 902 Organic matter 824 Crude protein (N6.25) 159 Starch 96 NNCCb 286 Crude fibre 162 Neutral-detergent fibre (NDF) 379 Acid-detergent fibre (ADF) 189 Acid-detergent lignin (ADL) 34
893 837 177 320 467 72 193 88 15
a Containing a coccidiostatic (66 mg of robenidineR/kg) and no antibiotics added. b Nonnitrogenous cellular content=(organic matter)-NDF-(crude protein).
Dry matter (DM) was analysed on feeds, faeces, and caecal content by heating at 103 8C for 24 h. Organic matter (OM) was determined by ashing samples at 550 8C for 5 h. Nitrogen was detected by DUMAS combustion method using a Leco apparatus (model FP-428, Leco, St. Joseph, MI, USA), converted to crude protein (CP) using the factor 6.25. Gross energy was measured by an adiabatic calorimeter (PARR Instrument, Moline, IL). Starch was enzymically measured by quantitative hydrolysis with glucoamylase (after gelatinisation and autoclaving), and the released glucose was then measured using
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the hexokinase (EC. 2.7.1.1)-glucose-6-phosphate dehydrogenase (NAD, EC1.1.1.49) system (Boehringer Mannheim). Different fractions of the fibre cell wall [neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL)] were analysed by the Van Soest method (Van Soest et al., 1991, EGRAN, 2001). Fibrolytic activity was assayed from bacteria, after their extraction from the caecal sample (Gidenne et al., 2002). Cellulolytic, xylanolytic, and pectinolytic activities were determined, respectively, on carboxymethylcellulose, wood xylose, and citrus pectin purified substrates by measuring the quantity of release reducing sugars (glucose, xylose, galacturonic acids, respectively) after 60 min at 39 8C of enzyme preparation (0.1 mL) with substrate (1 mL). The reaction was stopped by heating at 100 8C for 5 min. Reducing sugars were quantified spectrophotometrically at 410 nm by a p-hydroxybenzoic acid hydrazide method (Lever, 1977). The quantity of released sugars was expressed as micromole of reducing sugars per gram of dry matter of caecal content and per hour. Volatile fatty acids were extracted from caecal liquid digesta, and were measured by gas phase chromatography (P9000, Chrompack, Middelburg, The Netherlands) on semicapillary columns (Bellier, 1994). Ammonia concentration was analysed spectrophotometrically at 660 nm according to Verdow et al. (1977). 2.4. Statistical analyses For conventional rabbits, data of microbial activity (BFA, VFA) and caecal traits were analysed according to a bifactorial variance analysis, according to age (28, 42, 56, and 70 days) and diets (SF and FD), using the GLM procedure (SAS OnlineDoc.R, Release 8.01 for SunOs, SAS Institute, Cary, NC, USA). No significant interaction was detected between age and diet effect, and pooled data from
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SF and FD diets were presented in Figs. 1 and 2. The impact of the sanitary status (conventional vs. SPF) and interactions with diets was analysed on the caecal microbial activity of 70-day-old rabbits. As interaction among these two effects was significant, at 70 days of age, the data of bacterial fibrolytic activity were presented separately within each sanitary status (Fig. 3). For data of fermentation pattern, no significant interaction was detected among diet and sanitary status, and they were presented, first according to age and microbial status, and with pooled data of SF and FD groups for the age 70 days (Table 3), and second according to diet and sanitary status at 70 days of age (Table 4). Partial coefficients of correlation between different parameters were obtained through a multivariate analysis of variance. Bartlett tests were performed to analyse the homogeneity of variance, and when the variances were heterogeneous, statistical analyses were performed on the logn transformed variables. Data on digestibility measurements were analysed according to diet, sanitary status, and their interaction (Table 5).
3. Results 3.1. Growth and caecal development From 4 to 10 weeks old, the feed intake of healthy conventional rabbits was 31% lower for FD than for SF diet (mean=85 vs. 124 g/days), while it was only 21% lower in SPF animal (94 vs. 119 g/days; Pb0.01 for the interaction diet x status). Weight gain was significantly lower (12%) for conventional rabbits fed the FD diet compared to the SF one (37.7 vs. 43.0 g/days), whereas SPF had a slightly faster growth with the FD diet (41.9 vs. 39.7, Pb0.01 for the interaction diet x status). No significant interaction between diet and sanitary status was found for caecal development, either for the organ weight or its dry matter (Table 2). The
Fig. 1. Postweaning development of the caecal fermentation pattern in conventional rabbit. a, b: Means, between age, having a common superscript did not differ significantly at the level P=0.05. *Pooled data from SF and FD groups.
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Between 28 and 42 days, the bacterial fibrolytic activity decreased significantly by 32% and 60% for xylanase and CMCase, respectively (Fig. 2), while pectinase activity decreased more regularly from 28 to 70 days by 28%. 3.3. Microbial activity according to age and sanitary status
Fig. 2. Postweaning development of bacterial fibrolytic activity in the caecum of conventional rabbit. a, b: Means (FS.D.) between age, having a common superscript did not differ significantly at the level P=0.05. *Pooled data from SF and FD groups.
postweaning development of the caecum was not significantly affected by the diet. SPF animals had a higher relative development of the caecum (+10%, Pb0.01; Table 2) and a higher dry matter content (+1.6 units, Pb0.05) compared to conventional ones. 3.2. Microbial activity from weaning to 10 weeks old in conventional rabbits The caecal ammonia concentration decreased regularly from 28 until 70 days old (20%; Fig. 1), while total VFA (and acetate) concentration increased significantly by 46% between 42 and 56 days. The shape of tVFA curve was determined by the acetate, which was the predominant acid (84% to 76% of tVFA). In parallel, the caecal pH also decreased significantly between 42 and 56 days of age, from 6.12 to 5.67, and then remained steady. The fermentation profile also evolved with age: the molar proportion of acetate decreased linearly between 28 and 70 days (84% and 76%, respectively, Pb0.001), whereas that of butyric acid doubled, reaching 18% at 70 days. Because the proportion of propionate remained steady, the ratio C3/C4 was three times higher at weaning (0.62) compared to 70 days old (0.22). A negative correlation was found between pH and tVFA (r=0.67, n=89, Pb0.001), but not between the pH and ammonia ( P=0.28).
Significant interactions were detected between age and sanitary status for the tVFA level and the pH (Table 3). At weaning, the tVFA and C2 concentrations were similar between conventional and SPF rabbits (55.7 and 50.3 mM/L and 45.8 and 39.4 mM/L, respectively). But between 28 and 70 days, the concentration of tVFA, C2 and C3 decreased by 23%, 34%, and 47%, respectively, for SPF group, while the inverse situation was found in C group (+31% for tVFA, +22% for C2). Caecal pH followed the same trend: a significant decrease between 28 and 70 days old only for the C group. The changes in fermentation profile with age were similar among the sanitary status. However, the molar proportion of C4 was 27% higher in C group compared to SPF one, while the C3 proportion was lower by 33%, and the acetate proportion was not significantly affected. Because significant interactions were detected between the sanitary status and the diet for BFA criteria, data were analysed independently for C and SPF rabbits (Fig. 3), and then independently for SF and DF diets (at 70 days only). CMCase and xylanase activity was one to three times higher in conventional rabbits compared to SPF ones, while the pectinase activity did not differ among the two groups. 3.4. Effect of dietary fibre level No interaction was detected between sanitary and nutritional status at 70 days of age (except for BFA). Dry matter proportion and dry caecal content tended to be higher with the FD compared to the SF diet (+6%, P=0.11; +11%, P=0.10, respectively). The concentration of tVFA was 10% lower for the FD diet ( Pb0.05), and the ammonia concentration also tended to be higher for FD diet (Table 4). The concentration and the molar proportion of C2, respectively, reduced by 17% ( P=0.018) and by 8% ( Pb0.001) in DF compared to SF diet. Conversely, the molar proportion of the C4 was higher with the fibre-deficient diet. In 70-day-old rabbits (C or SPF group), pectinasic activity was significantly reduced by 30% when rabbits were fed the FD diet compared to the SF one (Fig. 3). Whole tract digestibility coefficients of organic matter or energy increased sharply when the level of the dietary fibre level decreased from SF to DF diet (Table 5). However, this increase was weaker in conventional compared to SPF rabbits (significant interaction among effects of diet and sanitary status). A similar interaction was found for the digestion of protein or fibre fractions. Few variations in fibre digestion were found in C rabbits between the two diets, whereas a higher fibre digestive efficiency was found
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Fig. 3. Effect of nutritional and sanitary status on bacterial fibrolytic activities in healthy growing rabbits. a, b: Means (F S.D.) between age within microbial status, having a common superscript did not differ significantly at the level P=0.05 ; A, B: means, between microbial status within age, having a common superscript did not differ significantly at the level P=0.05. At 70 days of age, interaction between diet and microbial status was significant ( Pb0.05) for CMCase and xylanase and within a diet, interaction between age and microbial status was significant ( Pb0.05) for pectinase. *Significant difference among diet ( Pb0.05) at 70 days (within microbial status).
in SPF rabbits fed the FD diet. Digestibility of NNCC constituents was similar among the two groups and was five units higher for FD diet.
4. Discussion 4.1. Postweaning development of the caecal microbial activity in the conventional rabbits Bacterial fermentation and fibrolytic activity were already well developed at 28 days of age, while the young were still consuming milk, as well as solid feed.
No interactions between microbial status and nutritional status were detected on the development of fibrolysis or fermentations. As previously observed (Bellier et al., 1995; Piattoni et al., 1995; Padilha et al., 1995; Xiccato et al., 2001), the proportion of butyrate exceeds that of propionate, and the ratio C3/C4 decreased with age. This particular fermentation profile originated in the composition of rabbit caecal flora of the rabbit (Adjiri et al., 1992), with BacteroRdes as a predominant bacterial genus. Nevertheless, butyrate is not a major product of BacteroRdes but a major product of Fusobacterium genus (Holdeman et al., 1984). Furthermore, Prohaszka and Szemeredi (1984) detected, in vitro, a high butyric
Table 2 Caecal development according to the sanitary status, in healthy growing rabbitsa Sanitary status
Organ weight (g) Organ (%LW) Content weight (g) Dry matter (%)
Age (days)
Statistical analysis
Conventional n=54
SPF n=30
28 n=41
70a n=43
rCV (%)
Sanitary status
Age
Inter action
21.3 1.42 73.8 24.1
27.9 1.60 98.1 25.7
8.6 1.32 23.0 25.1
38.0 1.61 138.3 24.2
14.4 14.0 25.5 9.6
ns ** ns **
*** *** *** *
y ns ns ns
n: Number of measurements; rCV, %: residual coefficient of variation; ns: not significant PN0.10; y: Pb0.10; *: Pb0.05; **; Pb0.01; ***: Pb0.001. a Pooled data of SF and FD groups.
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Table 3 Effect of sanitary status and age on fermentation parameters in healthy growing rabbits Sanitary status Conventional
SPF
Statistical analysis
Age (days)
28 n=30
70a n=24
28 n=12
70a n=18
rCV (%)
Age
Sanitary status
Interaction
pH NH3–N (mM/L) Total VFA (mM/L) C2 (%) C3 (%) C4 (%) C3/C4 ratio
6.10a 12.3a 55.7a 83.9a 6.4aA 9.5aA 0.83aA
5.70b 9.9b 73.0bA 76.2b 4.9bA 19.0b 0.22bA
6.20 12.6 50.3a 82.4a 9.9aB 6.7aB 2.26aB
6.18 10.6 37.9bB 76.0b 7.9bB 15.8b 0.55bB
4.1 20.9 24.0 5.6 35.9 31.8 95.9
*** ns *** ns *** ** ***
*** *** ns *** ** *** ***
*** ns *** ns ns ns **
rCV %, *, n, ns: see Table 2; a,b: means, between age within the same sanitary status, having a common superscript did not differ significantly at the level P=0.05; A, B: means, between sanitary status within the same age, having a common superscript did not differ significantly at the level P=0.05. a Pooled data of SF and FD groups.
production from Fusobacterium cultivated on a caecal substrate. Whatever the age, the pectinolytic activity was prevalent and was 4 to 15 times higher than xylanolytic or cellulolytic ones, as previously reported (Marounek et al., 1995; Gidenne et al., 2002). This hierarchy in fibrolytic activities also corresponds to that of digestive efficacy of the rabbit for cell wall constituents (pectinsNhemicellulosesNcellulose), and also to results of microbiological enumeration reporting higher counts of pectinolytic and hemicellulolytic bacteria compared to cellulolytic (Boulahrouf et al., 1991). In agreement with Gidenne et al. (2002), we found that fibrolytic activity was already present at 4 weeks of age. Thereafter, the fibrolysis potential was stable and even tended to decrease, particularly for pectinolytic activity. At weaning, the interindividual variations seemed higher for fermentation and fibrolytic activity. 4.2. Impact of sanitary status The particular sanitary status of SPF rabbits has been developed to study intestinal pathologies as coccidia (Coudert et al., 1978) or bRabbit Epizootic EnteropathyQ (Licois et al., 2000) and also to obtain a more standardised Table 4 Caecal fermentation pattern in healthy 70-day-old rabbits Diets
pH NH3–N (mM/L) Total VFA (mM/L) C2 (%) C3 (%) C4 (%) C3/C4 ratio
Statistical analysis
SF diet n=21
FD diet n=22
rCV (%)
Diet
Sanitary Status
Inter action
5.91 10.1
5.89 11.1
4.4 33.1
ns y
*** ***
ns ns
61.1
55.1
22.5
*
***
ns
79.1 6.1 14.8 0.40
73.1 5.3 20.2 0.30
5.4 19.3 42.2 39.6
*** ns * y
ns *** y ***
ns ns ns ns
rCV %, *, n, ns: see Table 2.
laboratory animal. However, this particular breeding environment would affect the digestion and more particularly the microbial activity. Indeed, when comparing previous studies using conventional or SPF animals (Padilha et al., 1995; Bellier and Gidenne, 1996), we found a 40% lower VFA concentration in the caecum for the latter. Present results confirmed our initial observation, and furthermore the microbial activity developed differently between weaning and 10 weeks of age among these two sanitary statuses: an increase of the fermentation level for conventional rabbits and a steadier pattern for SPF. The relatively low VFA concentration in 70-day-old SPF rabbits agreed with values found by Padilha et al. (1995) or with results from bgerm freeQ rabbits inoculated with a controlled caecal rabbit flora (Boot et al., 1985). Enzymatic activity of fibrolytic caecal bacteria of conventional rabbits exceeded that of SPF rabbits (except for pectinase), either at weaning or at 10 weeks of age. This was consistent with lower counts for cellulolytic flora in SPF rabbits (Padilha et al., 1995) compared to conventional (Gouet and Fonty, 1979; Boulahrouf et al., 1991). Moreover, Bennegadi et al. (2003) noticed a lower bacterial flora in SPF rabbits compared to conventional ones. The combination of a lower fibrolysis potential and a low fibrolytic bacterial population would explain the reduction of the fermentative activity in SPF rabbits, and also the lower fibre digestibility found for the standard fibre diet. It could be hypothesised that the caecal microflora would be less complex in SPF than in conventional rabbits leading to poorer interactions among the bacterial species, particularly for cell wall hydrolysis. Further researches are thus necessary to specify which bacterial populations are affected by the sanitary status, e.g., by using specific probes for fibrolytic caecal bacteria. 4.3. Effect of the dietary fibre level A sharp reduction of the fibre intake (23.4 vs. 7.5 g/ days of ADF, respectively, for SF and DF diets) did not affect the caecal development, but tended to decrease its
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Table 5 Effect of diet and sanitary status on nutrient digestion Sanitary status Conventional 1
SF diet n =9 Total tract apparent digestibility (%) Organic matter 66.1aA Energy 65.6aA Crude protein 75.6a NNCC 91.2 aA NDF 45.0A ADF 33.9A Cellulose 39.4 Hemicellulose 56.0A Nutritive value (air dry basis) Digestible energy (MJ/kg) 10.68 Digestible protein (g/kg) 121
FD diet n=8 80.4b 79.5b 82.7bA 96.3 bA 41.5 27.2 32.7 54.0
12.81 147
SPF
Statistical analysis1
SF diet n=10
FD diet n=10
61.6aB 61.0aB 72.9a 92.3aB 35.4aB 24.3aB 31.9 46.5aB
9.94 116
83.3b 82.7b 88.9 bB 97.7 bB 44.5b 33.4b 38.4 54.1 b
rCV (%)1
Sanitary status
Diet
Interaction
5.0 5.1 5.2 0.89 19.8 32.7 28.3 14.0
ns ns ns *** ns ns ns y
*** *** *** *** ns ns ns ns
** ** ** ns * * y y
13.47 158
a, b: Means between diets with the same sanitary status, having a common superscript did not differ significantly at the level P=0.05. A, B : Means between sanitary status with the same diet, having a common superscript did not differ significantly at the level P=0.05. 1 See Table 2.
water content, as previously reported (Pinheiro and Gidenne, 1999; Gidenne, 1995). Concurrently, the caecal dry matter level tended to be higher for with the FD diet than with SF diet. A low-fibre intake quantitatively and qualitatively modified the fermentative activity, with low VFA concentrations and higher butyrate molar proportion, as previously observed (Bellier and Gidenne, 1996; Gidenne et al., 2000). This lower fermentative activity was associated with a lower fibrolytic activity (particularly for pectins). It would be also attributable to a decrease in cellulolytic flora, as observed by Boulahrouf et al. (1991) for a lowfibre diet. The higher butyrate molar proportion found for animals having a low-fibre intake should be related with a higher proportion in BacteroR des flora found by Bennegadi et al. (2003) with the same fibre-deficient diet, because this bacterial genus would be responsible for butyrate production (Vernay and Marty, 1984). A classical improvement in diet digestibility was found when the dietary fibre level decreased. However, an interaction between the sanitary and the nutritional status was detected, because in conventional rabbits, the fibre digestibility was lower with fibre-deficient diet (Gidenne et al., 2000), while it was higher in SPF animals. This interaction was not reliable with variations in fermentative or fibrolytic activity.
5. Conclusion A direct role of the sanitary status on the caecal microbial activity was evidenced and was mainly expressed through a lower fermentative and fibrolytic activity in SPF rabbits. Few interactions between the nutritional status (fibre intake) and the sanitary status were observed, suggesting a similar adaptation of the fibrolytic flora to the level dietary fibre.
We also confirmed that the fibrolytic potential of the caecal ecosystem was developed already at weaning, although this maturation appeared less intensive for protected sanitary status such SPF.
Acknowledgement The authors thank P. Aymard, A. Lapanouse, and M. Segura (INRA Toulouse, Station de Recherches Cunicoles), and M. Dupuy, J.P. Molteni, and B. Sewald (INRA Tours, BASE) for their technical assistance.
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Boulahrouf, A., Fonty, G., Gouet, P., 1991. Establishment, counts and identification of the fibrolytic bacteria in the digestive tract of rabbit. Influence of feed cellulose content. Curr. Microbiol. 22, 1 – 25. Coudert, P., Vaissaire, J., Licois, D., 1978. Etude de l’e´volution de quelques parame` tres sanguins chez les lapereaux atteints de coccidioses intestinales. Rech. Me´d. Ve´t. 154, 437 – 440. Coudert, P., Licois, D., Besnard, J., 1988. Establishment of a specified pathogen free breeding colony (SPF) without hysterectomy and handrearing procedures. Proc. 4th Congress of WRSA, Budapest, Hungary, 10–14/10/88 vol. 2. , pp. 137 – 148. E.G.R.A.N., 2001. Technical note: attempts to harmonise chemical analyses of feeds and faeces, for rabbit feed evaluation. World Rabbit Sci. 9, 57 – 64. Gidenne, T., 1995. Effect of fibre level reduction and gluco-oligosaccharide addition on the growth performance and caecal fermentation in the growing rabbit. Anim. Feed Sci. Technol. 56, 253 – 263. Gidenne, T., 2003. Fibres in rabbit feeding for digestive troubles prevention: respective role of low-digested and digestible fibre. Livest. Prod. Sci. 81, 105 – 117. Gidenne, T., Pinheiro, V., Falca˜o E Cunha, L., 2000. A comprehensive approach of the rabbit digestion: consequences of a reduction in dietary fibre supply. Livest. Prod. Sci. 64, 225 – 237. Gidenne, T., Jehl, N., Segura, M., Michalet-Doreau, B., 2002. Microbial activity in the caecum of the rabbit around weaning: impact of a dietary fibre deficiency and of intake level. Anim. Feed Sci. Technol. 99, 107 – 118. Gouet, P., Fonty, G., 1979. Changes in the digestive microflora of holoxenic rabbits from birth until adulthood. Ann. Biol. Anim. Bioch. Biophys. 19, 553 – 566. Holdeman, L.V., Kelley, R.W., Moore, W.E.C., 1984. Genus I. Baceriodes. In: Krieg, N.R., Holt, J.G., (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 1. Williams and Wilkins, Baltimore, pp. 604 – 631. Lever, M., 1977. Carbohydrate determination with 4-hydroxybenzoic acid hydrazide (PAHhAH): effect of bismuth on the reaction. Anal. Biochem. 81, 21 – 27. Licois, D., Coudert, P., Ce´re´, N., Vautherot, J., 2000. Epizootic enterocolitis of the rabbit: review of current research. In: Blasco, A.Proc. 7th World
Rabbit Congress, 4–7 july 2000, vol. 2. Univ. Valenvia Publ., Valencia, Spain, pp. 299 – 306. Marounek, M., Vovk, S.J., Skrivanova, V., 1995. Distribution of activity of hydrolytic enzymes in the digestive tract of rabbits. Br. J. Nutr. 73, 463 – 469. Padilha, M.T.S., Licois, D., Gidenne, T., Carre´, B., Fonty, G., 1995. Relationships between microflora and caecal fermentation in rabbits before and after weaning. Reprod. Nutr. Dev. 35, 375 – 386. Perez, J.M., Lebas, F., Gidenne, T., Maertens, L., Xiccato, G., Parigi-Bini, R., Dalle Zotte, A., Cossu, M.E., Carazzolo, A., Villamide, M.J., Caraban˜o, R., Fraga, M.J., Ramos, M.A., Cervera, C., Blas, E., Fernandez Carmona, J., Falcao E Cunha, L., Bengala Freire, J., 1995. European reference method for in-vivo determination of diet digestibility in rabbits. World Rabbit Sci. 3, 41 – 43. Piattoni, F., Maertens, L., Demeyer, D., 1995. Age dependent variation of caecal contents composition of young rabbits. Arch. Anim. Nutr. 48, 34 – 355. Pinheiro, V., Gidenne, T., 1999. Impact of a fibre deficiency on zootechnical performances, caecal development, and composition of the ileal content in the growing rabbit. In: Perez, J.M. (Ed.), Proc. 8e`me Journ. Rech. Cunicole, 9–10 Nov. 1999. ITAVI publ., Paris, France, pp. 105 – 108. Prohaszka, L., Szemeredi, G., 1984. Regulation of the metabolism of volatile fatty acids in the caecum of rabbits. Zentralbl. Veterin7rmed. 31, 358 – 366. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and non starch polysaccharides in relation to animal. Nutr. J. Dairy Sci. 74, 3583 – 3597. Verdow, H., Van Echteld, C.J.A., Dekkers, E.M.J., 1977. Ammonia determination based on indophenol formation with sodium salicylate. Water Res. 12, 399 – 402. Vernay, M., Marty, J., 1984. Absorption and metabolism of butyric acid in rabbit hind gut. Comp. Biochem. Physiol. 77, 89 – 96. Xiccato, G., Trocino, A., Sartori, A., Queaque, P.I., 2001. Effect of age, early weaning and starter diet on the development of digestive organ and caecal fermentation in the young rabbit. In: Bolet, G. (Ed.), Proc. 9e`me Jour. Rech. Cunicole, 28–29 Nov. 2001. ITAVI publ., Paris, France, pp. 199 – 202.
Nutritional and sanitary statuses alter postweaning development of caecal microbial activity in the rabbit Nadia Bennegadi-Laurenta, Thierry Gidenneb,*, Dominique Licoisc a
ENSAT, De´partement des Sciences Animales, BP 107, 31326 Auzeville–Tolosane, Castanet-Tolosan Cedex, France b Station de Recherches Cunicoles, INRA Toulouse, Ch. de Borderouge, BP 27, 31326 Castanet-Tolosan, France c Bioagresseurs Sante´ Environnement, e´quipe pathologie cunicole, INRA Tours, 37380 Nouzilly, France Received 9 June 2004; received in revised form 3 September 2004; accepted 14 September 2004
Abstract The postweaning development of caecal microbial activity was studied in the rabbit according to the sanitary status (conventional bCQ vs. specified pathogen-free bSPFQ) and the nutritional status (standard-fibre bSFQ vs. deficient-fibre bDFQ diet). The two diets were distributed ad libitum from weaning (28 days) to 70 days of age, respectively, to 80 C and 72 SPF rabbits. From 28 to 42 days, the volatile fatty acids concentration in the caecum (tVFA) of C rabbits was 50 mM/L and increased by 46% between 42 and 56 days, without interactions with the diet effect. In parallel, the bacterial fibrolytic activity decreased for xylanase and CMCase (32% and 60%, respectively, Pb0.05), while pectinase activity decreased more regularly from 28 to 70 days (28%, Pb0.05). At weaning, tVFA was similar among C or SPF rabbits, while at 70 days, it decreased by 23% for SPF and increased in C group (+31%). Cellulasic and hemicellulasic activity of bacteria were two to three times lower, respectively, in SPF rabbits compared to conventional ones. No interaction was detected between sanitary and nutritional status at 70 days of age for the caecal fermentative activity. With the FD diet, tVFA decreased by 10%, while butyrate proportion increased by 37% (at 70 days), whatever the sanitary status. In 70-day-old rabbits (C or SPF group), pectinasic activity was reduced by 30% when rabbits were fed the FD compared to the SF one. D 2004 Elsevier Inc. All rights reserved. Keywords: Sanitary status; Caecal fermentation; Fibrolytic activity; Fibre deficiency; Young rabbit
1. Introduction The caecum of the rabbit is the main site of fermentative activity related to the presence of an abundant microbial flora (Gouet and Fonty, 1979), which develops significantly from 3 weeks of age, as the feed intake of the young rabbit shifts from milk to dry feed (Padilha et al., 1995). The flora development is however affected by the sanitary status, as shown by Bennegadi et al. (2003) who compared conventionally bred rabbits to those bred in a protected environment characterised by an absence of the main pathogenic * Corresponding author. Tel.: +33 5 61 28 51 03; fax: +33 5 61 28 53 19. E-mail address: [email protected] (T. Gidenne). 1095-6433/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2004.09.022
agents (specified pathogen-free bSPFQ; Coudert et al., 1988). Padilha et al. (1995) reported low caecal fermentation at weaning for SPF rabbits compared to previous studies on conventional rabbits. Bennegadi et al. (2001) reported that a dietary fibre deficiency favoured the occurrence of digestive troubles in the growing rabbit, but with a lower incidence for SPF animals compared to conventional ones. A role of caecal microbial activity has been hypothesised but not studied. Therefore, this study aimed to analyse the interactions between sanitary [SPF vs. conventional (C)] and nutritional statuses (diet with a standard-fibre vs. a fibre-deficient level) of the young rabbit on development of the caecal microbial activity assessed in three ways: fermentation end-products levels, bacterial fibrolytic activity, and fibre digestion.
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2. Material and methods 2.1. Animals, diets, and experimental design Two groups of hybrid rabbits (Oryctolagus cuniculus, strain INRA A1067) were bred according to two sanitary status, either in a conventional experimental unit (group C, n=80 rabbits) or in a highly protected environment (group SPF, n=72 rabbits). SPF environment is obtained according to the method of Coudert et al. (1988), that includes several criteria, such as ventilation with pressured air filtered at 10 Am, specific staff, and a regular confirmation that animals are free of coccidia, oxyuris, Pasteurella, Clostridium spiroforme and enteropathogenic Escherichia coli. Conventional environment is close to a standard breeding unit, with a closed ventilated and heated breeding room, but without microbiological control. Conventional animals were protected from coccidia as the feeds contain a coccidiostat (Table 1), and they had a low E. coli flora as shown in a previous microbiological study on the same rabbits (Bennegadi et al., 2003). From weaning (day 28) until 70 days old, rabbits were placed in separated rooms (conventional or SPF) and housed in individual wired cages (dimensions, 263365 cm, lhb) at room temperature (18F2 8C) with feedboxes and water pipes, a 12:12 light/dark cycle (lights on 07:00 h), and a minimum of 0.8 m3 air changes per hour. Before weaning, litters and does were fed a commercial diet. At weaning, C and SPF groups were allotted into two subgroups, according to the litter origin and weaning
Table 1 Ingredients and chemical composition of the experimental diets Diets Ingredients (%) Dehydrated alfalfa meal Dehydrated beet pulp Wheat bran Wheat Soya bean meal Wheat straw DL methionine Minerals Oligo element and vitaminsa
Control (SF) 30.0 20.0 20.0 12.4 10.0 6.0 0.2 0.9 0.5
Fibre deficient (FD) 9.0 7.5 5.0 54.3 19.0 2.0 0.2 2.5 0.5
weight, and they were fed ad libitum either a diet having a standard-fibre concentration (diet SF, 30 and 26 rabbits, respectively, for C and SPF groups) or a fibre-deficient diet (diet FD, 50 and 46 rabbits, respectively, for C and SPF groups). The two experimental diets were prepared in pelleted form and formulated to ensure no changes in fibre quality, but only in fibre quantity. For instance, the lignocellulosic content of the diet SF (189 g/kg) corresponded to current recommendations (Gidenne, 2003), while that of the FD diet was only 88 g/kg. The origin and proportions of fibre fractions were similar among diets, as well as the ratio of digestible protein/digestible energy (Table 1). Feeds and water were available ad libitum. 2.2. Measurements of digestive efficiency and microbial activity The digestive efficiency was measured on healthy conventional and SPF rabbits from 42 to 46 days with the two experimental diets (12 rabbits/diet/sanitary status) according to the European reference procedure (Perez et al., 1995). The caecal microbial activity was assessed through the analysis of fermentation end-products and of bacterial fibrolytic activity. Caecal contents were obtained after a lethal injection of anaesthetic solution (sodium thiopental) in the marginal ear vein at the end of the caecotrophy period (about 11:00 h), at 28, 42, 56, and 70 days in conventional rabbits (n=12/diet at each age), and at 28 and 70 days in SPF rabbits (n=12/diet at each age). The pH of the caecal digesta was taken immediately after laparotomy, with a glass electrode pH meter (pH 95, WTW, Weilherm, RFA). Portions of caecal digesta sample (1–3 g fresh matter) were collected in three different tubes containing, respectively, 10 mL of 0.025M of an anaerobic buffer MES-DTT, 2 ml of H3PO4 (2%, v/v), and 3 ml of H2SO4 (2%, v/v), which were storage solution for further analysis of bacterial fibrolytic activity bBFAQ (Gidenne et al., 2002), volatile fatty acids bVFAQ (Bellier, 1994), and NH3–N (Verdow et al., 1977), respectively. The portion of the caecal content for BFA analysis was stored at 80 8C and at 20 8C for VFA and NH3–N until analysis. The duration of the collection procedure did not exceed 15 min. 2.3. Biochemical analysis
Chemical analysis (g kg 1 air dry basis) Dry matter 902 Organic matter 824 Crude protein (N6.25) 159 Starch 96 NNCCb 286 Crude fibre 162 Neutral-detergent fibre (NDF) 379 Acid-detergent fibre (ADF) 189 Acid-detergent lignin (ADL) 34
893 837 177 320 467 72 193 88 15
a Containing a coccidiostatic (66 mg of robenidineR/kg) and no antibiotics added. b Nonnitrogenous cellular content=(organic matter)-NDF-(crude protein).
Dry matter (DM) was analysed on feeds, faeces, and caecal content by heating at 103 8C for 24 h. Organic matter (OM) was determined by ashing samples at 550 8C for 5 h. Nitrogen was detected by DUMAS combustion method using a Leco apparatus (model FP-428, Leco, St. Joseph, MI, USA), converted to crude protein (CP) using the factor 6.25. Gross energy was measured by an adiabatic calorimeter (PARR Instrument, Moline, IL). Starch was enzymically measured by quantitative hydrolysis with glucoamylase (after gelatinisation and autoclaving), and the released glucose was then measured using
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the hexokinase (EC. 2.7.1.1)-glucose-6-phosphate dehydrogenase (NAD, EC1.1.1.49) system (Boehringer Mannheim). Different fractions of the fibre cell wall [neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL)] were analysed by the Van Soest method (Van Soest et al., 1991, EGRAN, 2001). Fibrolytic activity was assayed from bacteria, after their extraction from the caecal sample (Gidenne et al., 2002). Cellulolytic, xylanolytic, and pectinolytic activities were determined, respectively, on carboxymethylcellulose, wood xylose, and citrus pectin purified substrates by measuring the quantity of release reducing sugars (glucose, xylose, galacturonic acids, respectively) after 60 min at 39 8C of enzyme preparation (0.1 mL) with substrate (1 mL). The reaction was stopped by heating at 100 8C for 5 min. Reducing sugars were quantified spectrophotometrically at 410 nm by a p-hydroxybenzoic acid hydrazide method (Lever, 1977). The quantity of released sugars was expressed as micromole of reducing sugars per gram of dry matter of caecal content and per hour. Volatile fatty acids were extracted from caecal liquid digesta, and were measured by gas phase chromatography (P9000, Chrompack, Middelburg, The Netherlands) on semicapillary columns (Bellier, 1994). Ammonia concentration was analysed spectrophotometrically at 660 nm according to Verdow et al. (1977). 2.4. Statistical analyses For conventional rabbits, data of microbial activity (BFA, VFA) and caecal traits were analysed according to a bifactorial variance analysis, according to age (28, 42, 56, and 70 days) and diets (SF and FD), using the GLM procedure (SAS OnlineDoc.R, Release 8.01 for SunOs, SAS Institute, Cary, NC, USA). No significant interaction was detected between age and diet effect, and pooled data from
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SF and FD diets were presented in Figs. 1 and 2. The impact of the sanitary status (conventional vs. SPF) and interactions with diets was analysed on the caecal microbial activity of 70-day-old rabbits. As interaction among these two effects was significant, at 70 days of age, the data of bacterial fibrolytic activity were presented separately within each sanitary status (Fig. 3). For data of fermentation pattern, no significant interaction was detected among diet and sanitary status, and they were presented, first according to age and microbial status, and with pooled data of SF and FD groups for the age 70 days (Table 3), and second according to diet and sanitary status at 70 days of age (Table 4). Partial coefficients of correlation between different parameters were obtained through a multivariate analysis of variance. Bartlett tests were performed to analyse the homogeneity of variance, and when the variances were heterogeneous, statistical analyses were performed on the logn transformed variables. Data on digestibility measurements were analysed according to diet, sanitary status, and their interaction (Table 5).
3. Results 3.1. Growth and caecal development From 4 to 10 weeks old, the feed intake of healthy conventional rabbits was 31% lower for FD than for SF diet (mean=85 vs. 124 g/days), while it was only 21% lower in SPF animal (94 vs. 119 g/days; Pb0.01 for the interaction diet x status). Weight gain was significantly lower (12%) for conventional rabbits fed the FD diet compared to the SF one (37.7 vs. 43.0 g/days), whereas SPF had a slightly faster growth with the FD diet (41.9 vs. 39.7, Pb0.01 for the interaction diet x status). No significant interaction between diet and sanitary status was found for caecal development, either for the organ weight or its dry matter (Table 2). The
Fig. 1. Postweaning development of the caecal fermentation pattern in conventional rabbit. a, b: Means, between age, having a common superscript did not differ significantly at the level P=0.05. *Pooled data from SF and FD groups.
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Between 28 and 42 days, the bacterial fibrolytic activity decreased significantly by 32% and 60% for xylanase and CMCase, respectively (Fig. 2), while pectinase activity decreased more regularly from 28 to 70 days by 28%. 3.3. Microbial activity according to age and sanitary status
Fig. 2. Postweaning development of bacterial fibrolytic activity in the caecum of conventional rabbit. a, b: Means (FS.D.) between age, having a common superscript did not differ significantly at the level P=0.05. *Pooled data from SF and FD groups.
postweaning development of the caecum was not significantly affected by the diet. SPF animals had a higher relative development of the caecum (+10%, Pb0.01; Table 2) and a higher dry matter content (+1.6 units, Pb0.05) compared to conventional ones. 3.2. Microbial activity from weaning to 10 weeks old in conventional rabbits The caecal ammonia concentration decreased regularly from 28 until 70 days old (20%; Fig. 1), while total VFA (and acetate) concentration increased significantly by 46% between 42 and 56 days. The shape of tVFA curve was determined by the acetate, which was the predominant acid (84% to 76% of tVFA). In parallel, the caecal pH also decreased significantly between 42 and 56 days of age, from 6.12 to 5.67, and then remained steady. The fermentation profile also evolved with age: the molar proportion of acetate decreased linearly between 28 and 70 days (84% and 76%, respectively, Pb0.001), whereas that of butyric acid doubled, reaching 18% at 70 days. Because the proportion of propionate remained steady, the ratio C3/C4 was three times higher at weaning (0.62) compared to 70 days old (0.22). A negative correlation was found between pH and tVFA (r=0.67, n=89, Pb0.001), but not between the pH and ammonia ( P=0.28).
Significant interactions were detected between age and sanitary status for the tVFA level and the pH (Table 3). At weaning, the tVFA and C2 concentrations were similar between conventional and SPF rabbits (55.7 and 50.3 mM/L and 45.8 and 39.4 mM/L, respectively). But between 28 and 70 days, the concentration of tVFA, C2 and C3 decreased by 23%, 34%, and 47%, respectively, for SPF group, while the inverse situation was found in C group (+31% for tVFA, +22% for C2). Caecal pH followed the same trend: a significant decrease between 28 and 70 days old only for the C group. The changes in fermentation profile with age were similar among the sanitary status. However, the molar proportion of C4 was 27% higher in C group compared to SPF one, while the C3 proportion was lower by 33%, and the acetate proportion was not significantly affected. Because significant interactions were detected between the sanitary status and the diet for BFA criteria, data were analysed independently for C and SPF rabbits (Fig. 3), and then independently for SF and DF diets (at 70 days only). CMCase and xylanase activity was one to three times higher in conventional rabbits compared to SPF ones, while the pectinase activity did not differ among the two groups. 3.4. Effect of dietary fibre level No interaction was detected between sanitary and nutritional status at 70 days of age (except for BFA). Dry matter proportion and dry caecal content tended to be higher with the FD compared to the SF diet (+6%, P=0.11; +11%, P=0.10, respectively). The concentration of tVFA was 10% lower for the FD diet ( Pb0.05), and the ammonia concentration also tended to be higher for FD diet (Table 4). The concentration and the molar proportion of C2, respectively, reduced by 17% ( P=0.018) and by 8% ( Pb0.001) in DF compared to SF diet. Conversely, the molar proportion of the C4 was higher with the fibre-deficient diet. In 70-day-old rabbits (C or SPF group), pectinasic activity was significantly reduced by 30% when rabbits were fed the FD diet compared to the SF one (Fig. 3). Whole tract digestibility coefficients of organic matter or energy increased sharply when the level of the dietary fibre level decreased from SF to DF diet (Table 5). However, this increase was weaker in conventional compared to SPF rabbits (significant interaction among effects of diet and sanitary status). A similar interaction was found for the digestion of protein or fibre fractions. Few variations in fibre digestion were found in C rabbits between the two diets, whereas a higher fibre digestive efficiency was found
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Fig. 3. Effect of nutritional and sanitary status on bacterial fibrolytic activities in healthy growing rabbits. a, b: Means (F S.D.) between age within microbial status, having a common superscript did not differ significantly at the level P=0.05 ; A, B: means, between microbial status within age, having a common superscript did not differ significantly at the level P=0.05. At 70 days of age, interaction between diet and microbial status was significant ( Pb0.05) for CMCase and xylanase and within a diet, interaction between age and microbial status was significant ( Pb0.05) for pectinase. *Significant difference among diet ( Pb0.05) at 70 days (within microbial status).
in SPF rabbits fed the FD diet. Digestibility of NNCC constituents was similar among the two groups and was five units higher for FD diet.
4. Discussion 4.1. Postweaning development of the caecal microbial activity in the conventional rabbits Bacterial fermentation and fibrolytic activity were already well developed at 28 days of age, while the young were still consuming milk, as well as solid feed.
No interactions between microbial status and nutritional status were detected on the development of fibrolysis or fermentations. As previously observed (Bellier et al., 1995; Piattoni et al., 1995; Padilha et al., 1995; Xiccato et al., 2001), the proportion of butyrate exceeds that of propionate, and the ratio C3/C4 decreased with age. This particular fermentation profile originated in the composition of rabbit caecal flora of the rabbit (Adjiri et al., 1992), with BacteroRdes as a predominant bacterial genus. Nevertheless, butyrate is not a major product of BacteroRdes but a major product of Fusobacterium genus (Holdeman et al., 1984). Furthermore, Prohaszka and Szemeredi (1984) detected, in vitro, a high butyric
Table 2 Caecal development according to the sanitary status, in healthy growing rabbitsa Sanitary status
Organ weight (g) Organ (%LW) Content weight (g) Dry matter (%)
Age (days)
Statistical analysis
Conventional n=54
SPF n=30
28 n=41
70a n=43
rCV (%)
Sanitary status
Age
Inter action
21.3 1.42 73.8 24.1
27.9 1.60 98.1 25.7
8.6 1.32 23.0 25.1
38.0 1.61 138.3 24.2
14.4 14.0 25.5 9.6
ns ** ns **
*** *** *** *
y ns ns ns
n: Number of measurements; rCV, %: residual coefficient of variation; ns: not significant PN0.10; y: Pb0.10; *: Pb0.05; **; Pb0.01; ***: Pb0.001. a Pooled data of SF and FD groups.
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Table 3 Effect of sanitary status and age on fermentation parameters in healthy growing rabbits Sanitary status Conventional
SPF
Statistical analysis
Age (days)
28 n=30
70a n=24
28 n=12
70a n=18
rCV (%)
Age
Sanitary status
Interaction
pH NH3–N (mM/L) Total VFA (mM/L) C2 (%) C3 (%) C4 (%) C3/C4 ratio
6.10a 12.3a 55.7a 83.9a 6.4aA 9.5aA 0.83aA
5.70b 9.9b 73.0bA 76.2b 4.9bA 19.0b 0.22bA
6.20 12.6 50.3a 82.4a 9.9aB 6.7aB 2.26aB
6.18 10.6 37.9bB 76.0b 7.9bB 15.8b 0.55bB
4.1 20.9 24.0 5.6 35.9 31.8 95.9
*** ns *** ns *** ** ***
*** *** ns *** ** *** ***
*** ns *** ns ns ns **
rCV %, *, n, ns: see Table 2; a,b: means, between age within the same sanitary status, having a common superscript did not differ significantly at the level P=0.05; A, B: means, between sanitary status within the same age, having a common superscript did not differ significantly at the level P=0.05. a Pooled data of SF and FD groups.
production from Fusobacterium cultivated on a caecal substrate. Whatever the age, the pectinolytic activity was prevalent and was 4 to 15 times higher than xylanolytic or cellulolytic ones, as previously reported (Marounek et al., 1995; Gidenne et al., 2002). This hierarchy in fibrolytic activities also corresponds to that of digestive efficacy of the rabbit for cell wall constituents (pectinsNhemicellulosesNcellulose), and also to results of microbiological enumeration reporting higher counts of pectinolytic and hemicellulolytic bacteria compared to cellulolytic (Boulahrouf et al., 1991). In agreement with Gidenne et al. (2002), we found that fibrolytic activity was already present at 4 weeks of age. Thereafter, the fibrolysis potential was stable and even tended to decrease, particularly for pectinolytic activity. At weaning, the interindividual variations seemed higher for fermentation and fibrolytic activity. 4.2. Impact of sanitary status The particular sanitary status of SPF rabbits has been developed to study intestinal pathologies as coccidia (Coudert et al., 1978) or bRabbit Epizootic EnteropathyQ (Licois et al., 2000) and also to obtain a more standardised Table 4 Caecal fermentation pattern in healthy 70-day-old rabbits Diets
pH NH3–N (mM/L) Total VFA (mM/L) C2 (%) C3 (%) C4 (%) C3/C4 ratio
Statistical analysis
SF diet n=21
FD diet n=22
rCV (%)
Diet
Sanitary Status
Inter action
5.91 10.1
5.89 11.1
4.4 33.1
ns y
*** ***
ns ns
61.1
55.1
22.5
*
***
ns
79.1 6.1 14.8 0.40
73.1 5.3 20.2 0.30
5.4 19.3 42.2 39.6
*** ns * y
ns *** y ***
ns ns ns ns
rCV %, *, n, ns: see Table 2.
laboratory animal. However, this particular breeding environment would affect the digestion and more particularly the microbial activity. Indeed, when comparing previous studies using conventional or SPF animals (Padilha et al., 1995; Bellier and Gidenne, 1996), we found a 40% lower VFA concentration in the caecum for the latter. Present results confirmed our initial observation, and furthermore the microbial activity developed differently between weaning and 10 weeks of age among these two sanitary statuses: an increase of the fermentation level for conventional rabbits and a steadier pattern for SPF. The relatively low VFA concentration in 70-day-old SPF rabbits agreed with values found by Padilha et al. (1995) or with results from bgerm freeQ rabbits inoculated with a controlled caecal rabbit flora (Boot et al., 1985). Enzymatic activity of fibrolytic caecal bacteria of conventional rabbits exceeded that of SPF rabbits (except for pectinase), either at weaning or at 10 weeks of age. This was consistent with lower counts for cellulolytic flora in SPF rabbits (Padilha et al., 1995) compared to conventional (Gouet and Fonty, 1979; Boulahrouf et al., 1991). Moreover, Bennegadi et al. (2003) noticed a lower bacterial flora in SPF rabbits compared to conventional ones. The combination of a lower fibrolysis potential and a low fibrolytic bacterial population would explain the reduction of the fermentative activity in SPF rabbits, and also the lower fibre digestibility found for the standard fibre diet. It could be hypothesised that the caecal microflora would be less complex in SPF than in conventional rabbits leading to poorer interactions among the bacterial species, particularly for cell wall hydrolysis. Further researches are thus necessary to specify which bacterial populations are affected by the sanitary status, e.g., by using specific probes for fibrolytic caecal bacteria. 4.3. Effect of the dietary fibre level A sharp reduction of the fibre intake (23.4 vs. 7.5 g/ days of ADF, respectively, for SF and DF diets) did not affect the caecal development, but tended to decrease its
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Table 5 Effect of diet and sanitary status on nutrient digestion Sanitary status Conventional 1
SF diet n =9 Total tract apparent digestibility (%) Organic matter 66.1aA Energy 65.6aA Crude protein 75.6a NNCC 91.2 aA NDF 45.0A ADF 33.9A Cellulose 39.4 Hemicellulose 56.0A Nutritive value (air dry basis) Digestible energy (MJ/kg) 10.68 Digestible protein (g/kg) 121
FD diet n=8 80.4b 79.5b 82.7bA 96.3 bA 41.5 27.2 32.7 54.0
12.81 147
SPF
Statistical analysis1
SF diet n=10
FD diet n=10
61.6aB 61.0aB 72.9a 92.3aB 35.4aB 24.3aB 31.9 46.5aB
9.94 116
83.3b 82.7b 88.9 bB 97.7 bB 44.5b 33.4b 38.4 54.1 b
rCV (%)1
Sanitary status
Diet
Interaction
5.0 5.1 5.2 0.89 19.8 32.7 28.3 14.0
ns ns ns *** ns ns ns y
*** *** *** *** ns ns ns ns
** ** ** ns * * y y
13.47 158
a, b: Means between diets with the same sanitary status, having a common superscript did not differ significantly at the level P=0.05. A, B : Means between sanitary status with the same diet, having a common superscript did not differ significantly at the level P=0.05. 1 See Table 2.
water content, as previously reported (Pinheiro and Gidenne, 1999; Gidenne, 1995). Concurrently, the caecal dry matter level tended to be higher for with the FD diet than with SF diet. A low-fibre intake quantitatively and qualitatively modified the fermentative activity, with low VFA concentrations and higher butyrate molar proportion, as previously observed (Bellier and Gidenne, 1996; Gidenne et al., 2000). This lower fermentative activity was associated with a lower fibrolytic activity (particularly for pectins). It would be also attributable to a decrease in cellulolytic flora, as observed by Boulahrouf et al. (1991) for a lowfibre diet. The higher butyrate molar proportion found for animals having a low-fibre intake should be related with a higher proportion in BacteroR des flora found by Bennegadi et al. (2003) with the same fibre-deficient diet, because this bacterial genus would be responsible for butyrate production (Vernay and Marty, 1984). A classical improvement in diet digestibility was found when the dietary fibre level decreased. However, an interaction between the sanitary and the nutritional status was detected, because in conventional rabbits, the fibre digestibility was lower with fibre-deficient diet (Gidenne et al., 2000), while it was higher in SPF animals. This interaction was not reliable with variations in fermentative or fibrolytic activity.
5. Conclusion A direct role of the sanitary status on the caecal microbial activity was evidenced and was mainly expressed through a lower fermentative and fibrolytic activity in SPF rabbits. Few interactions between the nutritional status (fibre intake) and the sanitary status were observed, suggesting a similar adaptation of the fibrolytic flora to the level dietary fibre.
We also confirmed that the fibrolytic potential of the caecal ecosystem was developed already at weaning, although this maturation appeared less intensive for protected sanitary status such SPF.
Acknowledgement The authors thank P. Aymard, A. Lapanouse, and M. Segura (INRA Toulouse, Station de Recherches Cunicoles), and M. Dupuy, J.P. Molteni, and B. Sewald (INRA Tours, BASE) for their technical assistance.
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