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Biodiversity and fermentative activity of caecal microbial communities in wild and farm rabbits from Spain
Anaerobe 18 (2012) 344e349
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Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Molecular biology, genetics and biotechnology
Biodiversity and fermentative activity of caecal microbial communities in wild and farm rabbits from Spain L. Abecia a, b, N. Rodríguez-Romero a, D.R. Yañez-Ruiz b, M. Fondevila a, * a b
Instituto Universitario de Investigación en Ciencias Ambientales, Dept. Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, M. Servet 177, 50013 Zaragoza, Spain Instituto de Nutrición Animal, Estación Experimental del Zaidín, CSIC, 18008 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2012 Received in revised form 26 March 2012 Accepted 16 April 2012 Available online 24 April 2012
In order to study the microbial caecal ecosystem of wild and domestic rabbits through the fermentation characteristics and concentration and diversity of bacterial and archaeal communities, caecal samples from sixteen wild rabbits (WR) were contrasted with two groups (n ¼ 4) of farm rabbits receiving low (LSF) or high (HSF) soluble fibre diets from 28 (weaning) to 51 days of age. DNA was extracted for quantifying bacteria and Archaea by qPCR and for biodiversity analysis of microbial communities by DGGE. Samples from WR had lower caecal pH and ammonia and higher volatile fatty acids concentration than farm animals. Lower acetate and higher butyrate proportions were detected in WR. Bacterial and archaeal DGGE profiles were clearly different between wild and farm rabbits, and diet-affected population of farm rabbits. Similarity index of bacteria was lower than 0.40 among WR, and 0.52 among farm rabbits. In conclusion, caecal fermentation characteristics differ between wild and farm rabbits, which harbour clearly different bacterial and archaeal communities. In farm rabbits, diversity is influenced by the dietary level of soluble fibre. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Microbial diversity Fermentation Wild rabbits Farm rabbits Caecum
1. Introduction In some Mediterranean countries, rabbits (Oryctolagus cuniculus) are commonly used for meat production under intensive practises. In Spain, meat breeds are slaughtered at around 60 days of age, with 2 kg live weight. Besides, there is also an important community of wild rabbits in the Iberian Peninsula, that is highlighted in the more than 5 million animals captured in 2008 [1]. Wild rabbits are lighter, reaching 1.5e2 kg in adulthood. The rabbit is a herbivorous species capable to ferment fibrous feeds in its caecum to provide energy by the presence of an abundant microbiota; however, the main role of this organ is recycling protein through intake of soft faeces, i.e. caecotrophy [2]. Feed intake in domestic rabbits is high, which ensures a rapid transit time [3]. Wild rabbits are herbivorous that maintain a selective behaviour: they choose diet according to local or seasonal availability [4,5], but they generally consume a high proportion of fibrous feeds [6], with starch sources having a lowand seasonal relevance, compared to domestic animals.
* Corresponding author. Tel.: þ34 876554171; fax: þ34 976761590. E-mail address: [email protected] (M. Fondevila). 1075-9964/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2012.04.004
The relative importance of some bacterial species in the caecum of farm rabbits has been studied by cultivation procedures [7,8], or using oligonucleotide probing methods [9,10]. In the last decade, attempts using molecular techniques have been also applied to the description of microbial biodiversity [11,12]. If it can be assumed that the microbial caecal ecosystem of the farm rabbit is not totally understood, even less is known about the digestive physiology of wild rabbits or the possible differences between these two type of animals, that can otherwise be expected because of either differences in dietary composition [13,14] or the level of intake [15]. Among other common microbial communities from the digestive tract, a community of methanogenic Archaea is present in the rabbit caecum, although there is not agreement about its importance [9,16]. Kusar and Avgustin [17] showed that the methanogen community of the rabbit caecum is unique and much less diverse than the rumen or the pig caecum, and suggested dominance by few species or strains from the genus Methanobrevibacter. The presence of protozoa has not been reported in the rabbit caecum either microscopically or by molecular techniques. This paper is a preliminary comparative study of the microbial communities of the caecum of wild and domestic rabbits, in terms of fermentative parameters and total concentration and biodiversity of bacteria and Archaea. Considering the importance of diet on caecal environment, samples of farm rabbits correspond to animals fed with different insoluble fibre proportions.
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2. Materials and methods
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measured at 260 nm with a NanoDrop (3.0.1, ND-1000, Willington, USA) and purity was accessed by measuring the A260/A280 ratio.
2.1. Experimental design All procedures were approved by the Comisión Ética Asesora en Experimentación Animal from the University of Zaragoza (Project License PI27/08). Care and use of animals were performed according to the Spanish Policy for Animal Protection RD1201/05, which meets the European Union Directive 86/609 on the protection of animals used for experimental and other scientific purposes. Sixteen wild rabbits (WR) that were living free close to the location of Magallón (Zaragoza, Spain; 41 500 N; 01 270 W) were shot by sport hunters under the supervision of the Servicio de Protección de la Naturaleza (Gobierno de Aragón) in February 2009. The region is characterised by a continental Mediterranean climate, with 12 and 3 C average maximum and minimum temperatures and 21 mm/m2 rainfall during the period of hunting. Animals (11 females and 5 males) came from 8 different barrows. In all cases, sampling was carried out immediately after slaughter (less than 15 min). Two groups of four White New Zealand rabbits (0.607 kg live weight) from the Servicio de Apoyo a la Experimentación Animal, University of Zaragoza, received two different commercial diets based on different proportions of neutral detergent soluble fibre (148 and 186 g/kg dry matter; LSF and HSF) from weaning (28 days of age) to 51 days. Diets were formulated to include 145 g and 370 g crude protein and neutral detergent fibre (NDF) per kg dry matter, respectively. Throughout the experiment, domestic rabbits were housed in a controlled environment room (18e24 C, 08:00e20:00 h light schedule). Farm rabbits (51 days of age, 1.522 kg) were slaughtered by cervical dislocation between 11:00 and 12:00 h. Once slaughtered, caecum of wild and domestic rabbits was excised, and the pH of caecal contents was measured directly inside the organ with a glass electrode pH-meter (CRISON 507, CRISON, Barcelona, Spain). Caecal contents (1 g) were immediately sampled, frozen in liquid nitrogen and stored at 80 C for subsequent microbiological analyses. Another 1 g sample was added to 1 ml of 18% formalin for optical counts of protozoa. In addition, two more samples (1 g) were taken and either acidified 1:1 with HCl 0.1 N or added with 2 ml distilled water and 1 ml of a 0.5 M H3PO4 and 0.05 M 4-metilvalerate solution, that were stored at 20 C for determination of ammonia and volatile fatty acid (VFA) concentration, respectively.
2.2. Analytical procedures Dry matter (DM) in feeds was determined by drying at 60 C to constant weight. Kjeldahl N was determined in a Kjeltec 2300 Ananlyzer Unit (Foss Tecator, Hoganas, Sweden), and results are given as crude protein (N 6.25). Concentration of neutral detergent fibre was determined according to [18] using an Ankom 220 Fibre Analyser equipment (Ankom Technology, New York); results are expressed exclusive of residual ash and aeamylase was used in the NDF analysis. Dietary content of NDSF was estimated according to [19]. Caecal ammonia concentration was determined colourimetrically as in [20]. Concentration of VFA was analysed by gas liquid chromatography, following the procedure described by [21] adapted to a capillary column J&W Scientific 19095F-123 HP-FFAP (30 m longitude and y 0.53 mm internal diameter), in an Agilent Plus 6890 series GC System. DNA was extracted from frozen samples using a QIAamp DNA Stool Mini Kit (QIAGEN Ltd, West Sussex, UK) following the manufacturer instructions, except that the samples were heated at 95 C for 5 min to lyse bacterial cells. DNA concentrations were
2.3. Denaturing gradient gel electrophoresis (DGGE) and real time PCR analysis DGGE analysis of bacterial microbial community was carried out as previously described [22], using the bacterial universal primers (positions 341e534) 50 -CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG-30 and 50 -ATT ACC GCG GCT GCT GG-30 . For the archaeal community, the mcrA gene were amplified by PCR using the primers forward 519f 50 -CAG CCG CCG CGG TAA-30 ; 915rGC reverse 50 -CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC CGT GCT CCC CCG CCA ATT CCT-30 [23]. The gels were scanned and the image was analysed with molecular analysis fingerprinting software (Quantity One e BIORAD Lab, Inc.) by scoring for the presence or absence of bands at different positions in each line. DGGE banding profiles were compared using Dice coefficient and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering algorithm and shown graphically as a dendrogram. The level of similarity was indicated by the percentage similarity coefficient bar located above each dendrogram. The Shannon index was used as a diversity index [24] and was calculated as follows: H ¼ S(pi)(ln pi), where pi is the ratio of one specific group of bacteria to the total microorganisms in the samples, and i is the total number of microbial species in the samples. Real time PCR was performed using an ABI PRISM 7000, Sequence Detection System using primers to target 16S rDNA total caecal bacteria: forward-50 -ACTCCTACGGGAGGCAG-30 ; reverse-50 GACTACCAGGGTATCTAATCC-30 [25]. Two microlitres of extracted DNA was added to amplification reaction (25 ml total volume) containing 0.2 ml of each primer, 12.5 ml of Master Mix Power SYBR Green qPCR of Applied Biosystems. Cycling condition for absolute quantification of total bacteria consisted of an initial hold to 95 C for 10 min followed by 40 cycles of 95 C for 15 s and 61 C for 1 min. The primer sets for detection and enumeration of Archaea were targeted against the methyl coenzyme-M reductase (mcrA) gene: forward-50 -TTCGGTGGATCDCARAGRGC-30 , reverse50 -GBARGTCGWAWCCGTAGAATCC-30 [26]. Real time PCR analyses were performed on iQ5 multicolour Real Time PCR Detection System (BioRad, Laboratories Inc., Hercules, CA, USA) using iQÔ SYBRÒ Green Supermix 2 Real Time PCR (BioRad), as described by [27]. Efficiency of amplifications was calculated using serial dilutions and they were accepted only when they were between 90 and 110%. 2.4. Statistical analysis Data were contrasted by one way ANOVA using the General Lineal Model (GLM) procedure of SAS [28], according to the model:
Xi ¼ m þ Ti þ εij where Ti represents the origin of rabbits (wild rabbits, n ¼ 12 or 16; farm rabbits given LSF, n ¼ 4; farm rabbits given HSF, n ¼ 4). LS means from the experimental treatments were compared by the least significant difference test, and differences among means with P < 0.05 and 0.05 < P < 0.10 were accepted as statistically significant differences and tendencies to differences, respectively. 3. Results Caecal pH, total ammonia concentration and total concentration and molar proportions of VFA are presented in Table 1. Valerate was
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Table 1 Caecal pH, ammonia concentration (mg/L) and volatile fatty acid (VFA) concentration (mM) and proportions in wild rabbits (WR, n ¼ 16) and farm rabbits given low (LS, n ¼ 4) and high (HS, n ¼ 4) levels of soluble fibre.
pH Ammonia VFA Acetate Propionate Butyrate Valerate
WR
LSF
HSF
SDa
Probability
5.69b 52.7b 119.4a 0.763b 0.052 0.180a 0.0036
6.15a 87.3a 87.0ab 0.840a 0.048 0.110b 0.0023
6.13a 73.9ab 84.5b 0.837a 0.045 0.103b 0.0032
0.349 20.45 29.63 0.0316 0.0134 0.0390 0.0025
0.027 0.023 0.049 <0.001 0.618 0.001 0.649
Within rows, different letters indicate significant differences among treatment means (P < 0.05). a SD, standard deviation.
detected in 11 out of 16 wild rabbits and in 7 out of 8 farm rabbits. Branched VFA (isobutyrate and isovalerate) were only detected in 4 wild rabbits, and therefore were not included in the table. No significant differences were observed between farm rabbits given either high or low dietary levels of soluble fibre in any studied parameter. Average caecal pH was lower in wild than farm rabbits irrespective of their diet (P ¼ 0.026), in agreement with their higher total VFA concentration (P ¼ 0.049). Molar proportion of acetate was lower, whereas that of butyrate was higher, in wild rabbits (P < 0.01), and no differences among treatments were detected in propionate and valerate proportions. Caecal ammonia concentration was also higher in wild than in farm rabbits (P ¼ 0.023). In this regard, it is worth mentioning that farm animals given LSF recorded a numerically higher ammonia concentration than HSF, although differences did not reached significance. Bacterial caecal profile of the caecum of wild and farm rabbits was compared by DGGE (Fig. 1), and the dendrogram of the relative similarities of samples is shown in Fig. 2. A total of 58 different bands were detected. One animal (WR 4) was clearly separated from the others. Among the rest, farm rabbits clustered separately, depending on the diet they received. Rabbits from either HSF or LSF showed a high internal similarity (indexes of 0.59 and 0.54,
Fig. 1. Comparison of bacterial DGGE profiles of the amplicons of caecal samples from wild (WR, n ¼ 16) and farm rabbits given diets with high (HSF, n ¼ 4) or low (LSF, n ¼ 4) content of soluble fibre.
Fig. 2. Dendrogram obtained from the DGGE analysis of the eubacterial community of caecal samples from wild (WR) and farm rabbits given diets with high (HSF) or low (LSF) content of soluble fibre. The scale shows the proportion of similarity.
respectively), and similarity index between diets was 0.52. Wild rabbits clustered separately, with a high dispersion among them, and a minor relationship with farm animals. Any effect of the litter, assumed as the rabbits coming from each barrow was not apparent. The DGGE gel and the dendrogram of UPGMA distances from the DGGE banding pattern of Archaea are shown in Figs. 3 and 4. A restriction on the numbers of samples was due to the comb size, and therefore only 12 wild rabbits were included in this analysis to obtain a DGGE with a proper resolution for the analyses. Up to 55
Fig. 3. Comparison of methanogenic DGGE profiles of the amplicons of caecal samples from wild (WR, n ¼ 12) and farm rabbits given diets with high (HSF, n ¼ 4) or low (LSF, n ¼ 4) content of soluble fibre.
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was also lowest in WR (P < 0.001), but in this case no differences due to the diet ingested in farm animals were detected. On average, 10.5 bands were detected from the DGGE analysis of Archaea community in WR, whereas among farm rabbits LSF showed more bands than HSF (21.8 and 11.3, respectively). Consequently, Evenness index were highest for LSF, and lowest for WR (P < 0.001), whereas Shannon index did not shown differences between HSF and WR, thus indicating a higher biodiversity in the Archaea community in farm rabbits given the former diet. 4. Discussion
Fig. 4. Dendrogram obtained from the DGGE analysis of the Archaeal community of caecal samples from wild (WR) and farm rabbits given diets with high (HSF) or low (LSF) content of soluble fibre. The scale shows the proportion of similarity.
different bands were detected in the gel, but whereas the average number in LSF was 21.8, it only reached 11.3 and 10.5 in HSF and WR, respectively. As it was observed for bacterial diversity, farm rabbits clustered together depending on their diet, reaching similarities of 0.50 and 0.53 for LSF and HSF, respectively. All wild rabbits were grouped in a different cluster, showing similarities below 0.40 with both groups of farm animals. Within-group diversity was higher in WR, and barrow did not show any apparent effect. Total concentration (ng DNA per mg fresh content) and microbial biodiversity estimated by the Shannon and Evenness indexes, for bacterial and archaeal communities in the rabbit caecum are shown in Table 2. Differences were detected in total concentration of bacteria (P < 0.001), being highest in farm rabbits given HSF, intermediate in LSF and lowest in WR. Average number of bands detected for LSF and HSF bacterial communities were 28.3 and 29.3, and there were no differences in biodiversity between farm rabbits despite the diet they fed. However, wild rabbits (18.4 bands) showed a lower diversity (P < 0.01). Archaeal total concentration
Table 2 Total concentration (ng DNA/mg fresh content) and Shannon and evenness indexes in bacterial and Archaea communities in the caecum of wild rabbits (WR, n ¼ 16 for bacteria and n ¼ 12 for Archaea) and farm rabbits given low (LS, n ¼ 4) and high (HS, n ¼ 4) levels of soluble fibre.
Bacteria Total concentration Shannon index Evenness index Archaea Total concentration Shannon index Evenness index
WR
LSF
HSF
SDa
Probability
15.719c 2.848b 0.676b
46.166b 3.339a 0.776a
71.441a 3.368a 0.830a
9.9679 0.3123 0.0744
<0.001 0.005 0.005
2.923b 2.332b 0.582c
13.394a 3.071a 0.792a
13.524a 2.397b 0.697b
4.4856 0.2082 0.0506
<0.001 <0.001 <0.001
Within rows, different letters indicate significant differences among treatment means (P < 0.05). a SD, standard deviation.
DGGE profiling was performed in order to obtain insight into the complexity of the microbial community of rabbit caecum, and to compare the community inhabiting fermentation sites of hosts from two different environments and feeding conditions, farm vs. wild rabbits. The presence of protozoa in caecal samples was not detected in any rabbit, and therefore no results are presented here. Previous studies in our laboratory [2] also failed to detect protozoa in caecal contents of rabbits, either by optical or molecular approaches. To our knowledge, there is no published report of the presence of protozoa in this host species. Whereas the protozoa might interact with archaeal community in the rumen and they are present in some hindgut fermentors such as the horse [29], this does not seem to be the case in the rabbit caecum. Feeding habits in wild rabbits are greatly conditioned by the distance to barrow, in order to prevent for predation [30], although this also depends on other factors such as feed abundance, predatory pressure or population density. Grasses high in structural polysaccharides are their main feed source [4] but, when feed is scarcely available, as it should be the case in this experiment (winter), rabbits tend to select the most nutritive parts of plants [4,5]. In any case, because of the season when the experiment was carried out the diet of wild rabbits should probably include high proportions of both NDF and soluble fibre, as well as a very low proportion of starch, in comparison with farm rabbits. Caecal environmental characteristics (Table 1) did not differ between farm rabbits, despite of their differences in soluble fibre contents. Both ammonia and total VFA concentration were in the upper range of previous observations [2,31], probably because of their high level of both soluble and insoluble fibre. In wild rabbits, caecal pH was more than 0.45 units lower than in farm ones, as a consequence of their high concentration of VFA that was 0.39 higher than that of farm animals. This should imply either a higher input of fermentable substrates reaching the caecum or an increased retention time of them into the organ. Besides, the VFA profile is characterised by a lower acetate and larger butyrate proportion in wild compared with farm rabbits. The capability of WR during winter to select vegetal parts rich in soluble sugars, that promote a higher butyrate concentration, rather than cell wall polysaccharides, that would increase the contribution of acetate, should help to explain these responses. The lower ammonia concentration in the caecum of WR could be associated to a lower protein input or a reduced proteolytic activity, since the lower microbial concentration in these animals discards a higher nitrogen utilisation by microbes. The lack of information about the feeding preferences of wild rabbits in terms of protein intake makes this question difficult to solve. The absence of differences in VFA caecal concentration between LSF and HSF despite the higher proportion of rapidly fermentable compounds in the latter is probably caused by an enhanced utilisation of available energy towards bacterial synthesis, which in fact is the major goal of caecum in rabbits [2], instead of fermentation itself. This also agrees with the lack of differences in abundance of Archaea between LSF and HSF, since
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the hydrogen required for methanogenesis comes from production of acetate and butyrate that did not differ among these treatments (Table 1). The fact that wild rabbits fed diets with high NDF [4,6] compared with farm rabbits partly explains the differences in total bacterial concentration in the caecum (Table 2), since the slow fermentability of NDF would contribute to impairing the rate of microbial protein synthesis [32]. Wallage-Drees and Deinum [4] suggest that, during winter time, wild rabbits may suffer an important shortage of energy intake, being even below the standards for maintenance. Besides, this low microbial concentration could also be related to a low protein input to the caecum in wild rabbits during the winter season, although the lack of information about their diet in this work does not allow supporting this thought. In contrast, differences in bacterial concentration among farm animals would depend on the level of soluble fibre, since a high input of highly digestible matter to the caecum in HSF increases bacterial growth [14,33]. Average proportion of DNA from Archaea in the caecal contents ranged from 0.16 to 0.22 of total microbial DNA, which is within the range observed by [9]. The presence of Archaea was detected in the caecum of all wild and farm rabbits. Belenguer et al. [31] observed a high variability in methane production in vivo, with only 2 out of 16 farm rabbits showing a significant concentration; however, these authors detected methane in all cases when caecal contents of such animals were incubated in vitro, thus indicating the presence of Archaea in all animals, even at low concentrations sometimes. It seems that methanogenic microorganisms in the large intestine must compete for H2 with others such as acetogenic and sulphate-reducing bacteria [34], the prevalence of one of these microbial groups probably depending on the rate of passage [35]. Further, methanogenic microorganisms are especially pH sensitive [36], and this parameter in WR was 0.45 units lower than in farm rabbits, which might explain the lower archaeal concentration in WR. In any case, the proportion of Archaea in WR compared to farm rabbits (0.22) was similar to that of bacteria (0.27). As it was reported recently for the rumen environment, the size of the methanogen population does not necessarily correlate with differences in feed efficiency, diet, or metabolic measurements [37]. Thus, the structure of the methanogenic community at the genus or species level may be more important for determining host feed efficiency under different dietary conditions [38]. The number of bands in the DGGE gel for Archaea detected in our study was 10e11 for both WR and HSF (Fig. 3), with is in the line of the scarce diversity of Archaea detected by [17] who reported lower methanogen diversity in the rabbit caecum compared with the fermentation compartments of ruminants and pigs. However, when rabbit caecum samples from this experiment were compared to others from rumen or faeces from goats [39], it was observed that rabbits hold a microbial community of similar complexity than that observed in ruminants. This fact might be due to the specificity of the set of primers used, 519f/915rGC, that was reported to be more suitable for studying the methanogenic archaeal community in the rumen in a comparison of nine primers set. Kusar and Avgustin [17] also indicated that the Archaea community of the rabbit caecum is unique and different to that of the rumen or pig faeces. They stated that one species of the genus Methanobrevibacter may dominate, as it has also been suggested by [40] in chickens. However, the number of bands was almost two-fold (22 bands) when farm rabbits were given LSF. This is difficult to explain, since NDF content was the same in both LSF and HSF diets, and fermentation parameters were almost identical (Table 1) but only two bands were present in all samples. In any case [41], suggest that there is large diversity in the methanogenic digestive community among individuals of the same species, depending on their location and feeding conditions.
The reported methanogenic PCR-DGGE profiles that have been detected in the rumen are greatly affected by diet [38]. Between low- and high-energy diets, the major pattern changed from a community containing predominantly Methanobrevibacter ruminantium NT7 in the first case to another where Methanobrevibacter smithii, Methanobrevibacter sp. AbM4, and/or M. ruminantium NT7 predominates in the latter. For each diet, the methanogenic PCRDGGE pattern was strongly associated with the host feed efficiency. 5. Conclusions Caecal fermentation characteristics differ between wild and farm rabbits, despite the level and kind of dietary fibre, thus highlighting differences in dietary habits. In a similar sense, these types of rabbits harbour clearly different bacterial and archaeal communities. It is worth noting that, despite the intra-group similarity, the number of archaeal species in the caecum of wild rabbits was close to that of farm rabbits given high-soluble fibre diets, which may indicate a selective feeding in the former. In farm rabbits, diversity is influenced by the dietary level of soluble fibre. To our knowledge, this is the first approach to the study of digestive ecosystem of wild rabbits, and its comparison with farm rabbits might directly explain the effect of environment and dietary habits. Acknowledgements This work was financed through the Projects AGL 2006-07596 from the Ministerio de Educación y Ciencia (Spain). The stage of Ms. Norelys Rodríguez-Romero in the University of Zaragoza was financed though a Doctoral fellowship from the Universidad Nacional Experimental del Táchira (Venezuela). Dr. L. Abecia acknowledges the receipt of a research contract from the Spanish National Research Council (CSIC, JAE-Doc Programme). References [1] MARM. Anuario de Estadística 2010. Medio natural: caza y pesca fluvial (cap. 12.5). Medio Rural y Marino, Gobierno de España: Ministerio de Agricultura, http://www.marm.es/estadistica/pags/anuario/2010; 2011 [last accessed 15/12/2011]. [2] Belenguer A, Balcells J, Fondevila M, Torre C. Caecotrophes intake in growing rabbits estimated either from urinary excretion of purine derivatives or from direct measurement using animals provided with a neck collar: effect of type and level of dietary carbohydrate. Anim Sci 2002;74:135e44. [3] Gidenne T, Pérez JM. Effect of dietary starch origin on digestion in the rabbit. 2. Starch hydrolysis in the small intestine, cell wall degradation and rate of passage measurements. Anim Feed Sci Technol 1993;42:249e57. [4] Wallage-Drees JM, Deinum B. Quality of the diet selected by wild rabbits (Oryctolagus cuniculus L.) in autumn and winter. Neth J Zool 1986;36:438e48. [5] Martins H, Milne JA, Rego F. Seasonal and spatial variation in the diet of the wild rabbit (Oryctolagus cuniculus) in Portugal. J Zool. London 2002;258: 395e404. [6] Soriguer RC. Alimentación del conejo (Oryctolagus cuniculus L.) en Doñana, SO España. Doñana, Acta Vertebrata 1988;15:141e50. [7] Boulahrouf A, Fonty G, Gouet P. Establishment, counts and identification of the fibrolytic bacteria in the digestive tract of rabbit. Influence of feed cellulose content. Curr Microbiol 1991;22:1e25. [8] Padilha MTS, Licois D, Gidenne T, Carre B, Fonty G. Relationships between microflora and cecal fermentation in rabbits before and after weaning. Reprod Nutr Develop 1995;35:375e86. [9] Bennegadi N, Fonty G, Millet L, Gidenne T, Licois D. Effects of age and dietary fibre level on caecal microbial communities of conventional and specific pathogen-free rabbits. Microb Ecol Health Dis 2003;15:23e32. [10] Sirotek K, Marounek M, Rada V, Benda V. Isolation and characterization of rabbit caecal pectinolytic bacteria. Folia Microbiol 2001;46:79e82. [11] Abecia L, Fondevila M, Balcells J, Edwards JE, Newbold CJ, McEwan NR. Molecular profiling of bacterial species in the rabbit caecum. FEMS Microbiol Lett 2005;244:111e5. [12] Monteils V, Cauquil L, Combes S, Godon JJ, Gidenne T. Potential core species and satellite species in the bacterial community within the rabbit caecum. FEMS Microbiol Ecol 2008;66:620e9.
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[28] SAS Institute. SAS/STAT users guide, (release 9.2). Cary, NC, USA: SAS Inst. Inc; 2008. [29] Egan CE, Snelling TJ, McEwan NR. The onset of ciliate populations in newborn foals. Acta Protozool 2010;49:145e7. [30] Stott P. Use of space by sympatric European hares (Lepus europaeus) and European rabbits (Oryctolagus cuniculus) in Australia. Mamm Biol 2003;68: 317e27. [31] Belenguer A, Fondevila M, Balcells J, Abecia L, Lachica M, Carro MD. Methanogenesis in rabbit caecum as affected by the fermentation pattern: in vitro and in vivo measurements. World Rabbit Sci 2011;19:75e83. [32] García J, Carabaño R, Pérez-Alba L, de Blas JC. Effect of fiber source on cecal fermentation and nitrogen recycled through cecotrophy in rabbits. J Anim Sci 2000;78:638e46. [33] Gidenne T, Jehl N, Lapanouse A, Segura M. Inter-relationship of microbial activity, digestion and gut health in the rabbit: effect of substituting fibre by starch in diets having a high proportion of rapidly fermentable polysaccharides. Br J Nutr 2004;92:95e104. [34] de Graeve KG, Grivet JP, Durand M, Beaumatin P, Cordelet C, Hannequart G, et al. Competition between reductive acetogenesis and methanogenesis in the pig large-intestinal flora. J Appl Bacteriol 1994;76:55e61. [35] Morvan B, Bonnemoy F, Fonty G, Gouet P. Quantitative determination of H2utilizing acetogenic and sulphate-reducing bacteria and methanogenic archaea from digestive tract of different mammals. Curr Microbiol 1996;32: 129e33. [36] Russell JB. The importance of pH in the regulation of ruminal acetate to propionate ratio and methane production in vitro. J Dairy Sci 1998;81: 3222e30. [37] Mosoni P, Martin C, Forano E, Morgavi DP. Long-term defaunation increases the abundance of cellulolytic ruminococci and methanogens, but does not affect the bacterial and methanogen diversity in the rumen of sheep. J Anim Sci 2011;89:783e91. [38] Zhou M, Hernandez-Sanabria E, Guan LL. Characterization of variation in rumen methanogenic communities under different dietary and host feed efficiency conditions, as determined by PCR-denaturing gradient gel electrophoresis analysis. Appl Environ Microbiol 2010;76:3776e86. [39] Abecia L, Fondevila M, Rodriguez-Romero N, Martínez G, Balcells J, YañezRuiz DR. Methanogen community structure in fermentation compartments of goats and wild and domestic rabbits. In: Proc. 15th ESVCN Congress. Spain: Zaragoza; 2011. p. 143. [40] Saengkerdsub S, Anderson RC, Wilkinson HH, Kim W-K, Nisbet DJ, Ricke SC. Identification and quantification of methanogenic archaea in adult chicken ceca. Appl Environ Microbiol 2007;73:353e6. [41] Turnbull KL, Smith RP, St-Pierre B, Wright AD. Molecular diversity of methanogens in fecal samples from Bactrian camels (Camelus bactrianus) at two zoos. Res Vet Sci, in press (available online 12 Sept 2011).
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Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Molecular biology, genetics and biotechnology
Biodiversity and fermentative activity of caecal microbial communities in wild and farm rabbits from Spain L. Abecia a, b, N. Rodríguez-Romero a, D.R. Yañez-Ruiz b, M. Fondevila a, * a b
Instituto Universitario de Investigación en Ciencias Ambientales, Dept. Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, M. Servet 177, 50013 Zaragoza, Spain Instituto de Nutrición Animal, Estación Experimental del Zaidín, CSIC, 18008 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2012 Received in revised form 26 March 2012 Accepted 16 April 2012 Available online 24 April 2012
In order to study the microbial caecal ecosystem of wild and domestic rabbits through the fermentation characteristics and concentration and diversity of bacterial and archaeal communities, caecal samples from sixteen wild rabbits (WR) were contrasted with two groups (n ¼ 4) of farm rabbits receiving low (LSF) or high (HSF) soluble fibre diets from 28 (weaning) to 51 days of age. DNA was extracted for quantifying bacteria and Archaea by qPCR and for biodiversity analysis of microbial communities by DGGE. Samples from WR had lower caecal pH and ammonia and higher volatile fatty acids concentration than farm animals. Lower acetate and higher butyrate proportions were detected in WR. Bacterial and archaeal DGGE profiles were clearly different between wild and farm rabbits, and diet-affected population of farm rabbits. Similarity index of bacteria was lower than 0.40 among WR, and 0.52 among farm rabbits. In conclusion, caecal fermentation characteristics differ between wild and farm rabbits, which harbour clearly different bacterial and archaeal communities. In farm rabbits, diversity is influenced by the dietary level of soluble fibre. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Microbial diversity Fermentation Wild rabbits Farm rabbits Caecum
1. Introduction In some Mediterranean countries, rabbits (Oryctolagus cuniculus) are commonly used for meat production under intensive practises. In Spain, meat breeds are slaughtered at around 60 days of age, with 2 kg live weight. Besides, there is also an important community of wild rabbits in the Iberian Peninsula, that is highlighted in the more than 5 million animals captured in 2008 [1]. Wild rabbits are lighter, reaching 1.5e2 kg in adulthood. The rabbit is a herbivorous species capable to ferment fibrous feeds in its caecum to provide energy by the presence of an abundant microbiota; however, the main role of this organ is recycling protein through intake of soft faeces, i.e. caecotrophy [2]. Feed intake in domestic rabbits is high, which ensures a rapid transit time [3]. Wild rabbits are herbivorous that maintain a selective behaviour: they choose diet according to local or seasonal availability [4,5], but they generally consume a high proportion of fibrous feeds [6], with starch sources having a lowand seasonal relevance, compared to domestic animals.
* Corresponding author. Tel.: þ34 876554171; fax: þ34 976761590. E-mail address: [email protected] (M. Fondevila). 1075-9964/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2012.04.004
The relative importance of some bacterial species in the caecum of farm rabbits has been studied by cultivation procedures [7,8], or using oligonucleotide probing methods [9,10]. In the last decade, attempts using molecular techniques have been also applied to the description of microbial biodiversity [11,12]. If it can be assumed that the microbial caecal ecosystem of the farm rabbit is not totally understood, even less is known about the digestive physiology of wild rabbits or the possible differences between these two type of animals, that can otherwise be expected because of either differences in dietary composition [13,14] or the level of intake [15]. Among other common microbial communities from the digestive tract, a community of methanogenic Archaea is present in the rabbit caecum, although there is not agreement about its importance [9,16]. Kusar and Avgustin [17] showed that the methanogen community of the rabbit caecum is unique and much less diverse than the rumen or the pig caecum, and suggested dominance by few species or strains from the genus Methanobrevibacter. The presence of protozoa has not been reported in the rabbit caecum either microscopically or by molecular techniques. This paper is a preliminary comparative study of the microbial communities of the caecum of wild and domestic rabbits, in terms of fermentative parameters and total concentration and biodiversity of bacteria and Archaea. Considering the importance of diet on caecal environment, samples of farm rabbits correspond to animals fed with different insoluble fibre proportions.
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2. Materials and methods
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measured at 260 nm with a NanoDrop (3.0.1, ND-1000, Willington, USA) and purity was accessed by measuring the A260/A280 ratio.
2.1. Experimental design All procedures were approved by the Comisión Ética Asesora en Experimentación Animal from the University of Zaragoza (Project License PI27/08). Care and use of animals were performed according to the Spanish Policy for Animal Protection RD1201/05, which meets the European Union Directive 86/609 on the protection of animals used for experimental and other scientific purposes. Sixteen wild rabbits (WR) that were living free close to the location of Magallón (Zaragoza, Spain; 41 500 N; 01 270 W) were shot by sport hunters under the supervision of the Servicio de Protección de la Naturaleza (Gobierno de Aragón) in February 2009. The region is characterised by a continental Mediterranean climate, with 12 and 3 C average maximum and minimum temperatures and 21 mm/m2 rainfall during the period of hunting. Animals (11 females and 5 males) came from 8 different barrows. In all cases, sampling was carried out immediately after slaughter (less than 15 min). Two groups of four White New Zealand rabbits (0.607 kg live weight) from the Servicio de Apoyo a la Experimentación Animal, University of Zaragoza, received two different commercial diets based on different proportions of neutral detergent soluble fibre (148 and 186 g/kg dry matter; LSF and HSF) from weaning (28 days of age) to 51 days. Diets were formulated to include 145 g and 370 g crude protein and neutral detergent fibre (NDF) per kg dry matter, respectively. Throughout the experiment, domestic rabbits were housed in a controlled environment room (18e24 C, 08:00e20:00 h light schedule). Farm rabbits (51 days of age, 1.522 kg) were slaughtered by cervical dislocation between 11:00 and 12:00 h. Once slaughtered, caecum of wild and domestic rabbits was excised, and the pH of caecal contents was measured directly inside the organ with a glass electrode pH-meter (CRISON 507, CRISON, Barcelona, Spain). Caecal contents (1 g) were immediately sampled, frozen in liquid nitrogen and stored at 80 C for subsequent microbiological analyses. Another 1 g sample was added to 1 ml of 18% formalin for optical counts of protozoa. In addition, two more samples (1 g) were taken and either acidified 1:1 with HCl 0.1 N or added with 2 ml distilled water and 1 ml of a 0.5 M H3PO4 and 0.05 M 4-metilvalerate solution, that were stored at 20 C for determination of ammonia and volatile fatty acid (VFA) concentration, respectively.
2.2. Analytical procedures Dry matter (DM) in feeds was determined by drying at 60 C to constant weight. Kjeldahl N was determined in a Kjeltec 2300 Ananlyzer Unit (Foss Tecator, Hoganas, Sweden), and results are given as crude protein (N 6.25). Concentration of neutral detergent fibre was determined according to [18] using an Ankom 220 Fibre Analyser equipment (Ankom Technology, New York); results are expressed exclusive of residual ash and aeamylase was used in the NDF analysis. Dietary content of NDSF was estimated according to [19]. Caecal ammonia concentration was determined colourimetrically as in [20]. Concentration of VFA was analysed by gas liquid chromatography, following the procedure described by [21] adapted to a capillary column J&W Scientific 19095F-123 HP-FFAP (30 m longitude and y 0.53 mm internal diameter), in an Agilent Plus 6890 series GC System. DNA was extracted from frozen samples using a QIAamp DNA Stool Mini Kit (QIAGEN Ltd, West Sussex, UK) following the manufacturer instructions, except that the samples were heated at 95 C for 5 min to lyse bacterial cells. DNA concentrations were
2.3. Denaturing gradient gel electrophoresis (DGGE) and real time PCR analysis DGGE analysis of bacterial microbial community was carried out as previously described [22], using the bacterial universal primers (positions 341e534) 50 -CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG-30 and 50 -ATT ACC GCG GCT GCT GG-30 . For the archaeal community, the mcrA gene were amplified by PCR using the primers forward 519f 50 -CAG CCG CCG CGG TAA-30 ; 915rGC reverse 50 -CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC CGT GCT CCC CCG CCA ATT CCT-30 [23]. The gels were scanned and the image was analysed with molecular analysis fingerprinting software (Quantity One e BIORAD Lab, Inc.) by scoring for the presence or absence of bands at different positions in each line. DGGE banding profiles were compared using Dice coefficient and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering algorithm and shown graphically as a dendrogram. The level of similarity was indicated by the percentage similarity coefficient bar located above each dendrogram. The Shannon index was used as a diversity index [24] and was calculated as follows: H ¼ S(pi)(ln pi), where pi is the ratio of one specific group of bacteria to the total microorganisms in the samples, and i is the total number of microbial species in the samples. Real time PCR was performed using an ABI PRISM 7000, Sequence Detection System using primers to target 16S rDNA total caecal bacteria: forward-50 -ACTCCTACGGGAGGCAG-30 ; reverse-50 GACTACCAGGGTATCTAATCC-30 [25]. Two microlitres of extracted DNA was added to amplification reaction (25 ml total volume) containing 0.2 ml of each primer, 12.5 ml of Master Mix Power SYBR Green qPCR of Applied Biosystems. Cycling condition for absolute quantification of total bacteria consisted of an initial hold to 95 C for 10 min followed by 40 cycles of 95 C for 15 s and 61 C for 1 min. The primer sets for detection and enumeration of Archaea were targeted against the methyl coenzyme-M reductase (mcrA) gene: forward-50 -TTCGGTGGATCDCARAGRGC-30 , reverse50 -GBARGTCGWAWCCGTAGAATCC-30 [26]. Real time PCR analyses were performed on iQ5 multicolour Real Time PCR Detection System (BioRad, Laboratories Inc., Hercules, CA, USA) using iQÔ SYBRÒ Green Supermix 2 Real Time PCR (BioRad), as described by [27]. Efficiency of amplifications was calculated using serial dilutions and they were accepted only when they were between 90 and 110%. 2.4. Statistical analysis Data were contrasted by one way ANOVA using the General Lineal Model (GLM) procedure of SAS [28], according to the model:
Xi ¼ m þ Ti þ εij where Ti represents the origin of rabbits (wild rabbits, n ¼ 12 or 16; farm rabbits given LSF, n ¼ 4; farm rabbits given HSF, n ¼ 4). LS means from the experimental treatments were compared by the least significant difference test, and differences among means with P < 0.05 and 0.05 < P < 0.10 were accepted as statistically significant differences and tendencies to differences, respectively. 3. Results Caecal pH, total ammonia concentration and total concentration and molar proportions of VFA are presented in Table 1. Valerate was
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Table 1 Caecal pH, ammonia concentration (mg/L) and volatile fatty acid (VFA) concentration (mM) and proportions in wild rabbits (WR, n ¼ 16) and farm rabbits given low (LS, n ¼ 4) and high (HS, n ¼ 4) levels of soluble fibre.
pH Ammonia VFA Acetate Propionate Butyrate Valerate
WR
LSF
HSF
SDa
Probability
5.69b 52.7b 119.4a 0.763b 0.052 0.180a 0.0036
6.15a 87.3a 87.0ab 0.840a 0.048 0.110b 0.0023
6.13a 73.9ab 84.5b 0.837a 0.045 0.103b 0.0032
0.349 20.45 29.63 0.0316 0.0134 0.0390 0.0025
0.027 0.023 0.049 <0.001 0.618 0.001 0.649
Within rows, different letters indicate significant differences among treatment means (P < 0.05). a SD, standard deviation.
detected in 11 out of 16 wild rabbits and in 7 out of 8 farm rabbits. Branched VFA (isobutyrate and isovalerate) were only detected in 4 wild rabbits, and therefore were not included in the table. No significant differences were observed between farm rabbits given either high or low dietary levels of soluble fibre in any studied parameter. Average caecal pH was lower in wild than farm rabbits irrespective of their diet (P ¼ 0.026), in agreement with their higher total VFA concentration (P ¼ 0.049). Molar proportion of acetate was lower, whereas that of butyrate was higher, in wild rabbits (P < 0.01), and no differences among treatments were detected in propionate and valerate proportions. Caecal ammonia concentration was also higher in wild than in farm rabbits (P ¼ 0.023). In this regard, it is worth mentioning that farm animals given LSF recorded a numerically higher ammonia concentration than HSF, although differences did not reached significance. Bacterial caecal profile of the caecum of wild and farm rabbits was compared by DGGE (Fig. 1), and the dendrogram of the relative similarities of samples is shown in Fig. 2. A total of 58 different bands were detected. One animal (WR 4) was clearly separated from the others. Among the rest, farm rabbits clustered separately, depending on the diet they received. Rabbits from either HSF or LSF showed a high internal similarity (indexes of 0.59 and 0.54,
Fig. 1. Comparison of bacterial DGGE profiles of the amplicons of caecal samples from wild (WR, n ¼ 16) and farm rabbits given diets with high (HSF, n ¼ 4) or low (LSF, n ¼ 4) content of soluble fibre.
Fig. 2. Dendrogram obtained from the DGGE analysis of the eubacterial community of caecal samples from wild (WR) and farm rabbits given diets with high (HSF) or low (LSF) content of soluble fibre. The scale shows the proportion of similarity.
respectively), and similarity index between diets was 0.52. Wild rabbits clustered separately, with a high dispersion among them, and a minor relationship with farm animals. Any effect of the litter, assumed as the rabbits coming from each barrow was not apparent. The DGGE gel and the dendrogram of UPGMA distances from the DGGE banding pattern of Archaea are shown in Figs. 3 and 4. A restriction on the numbers of samples was due to the comb size, and therefore only 12 wild rabbits were included in this analysis to obtain a DGGE with a proper resolution for the analyses. Up to 55
Fig. 3. Comparison of methanogenic DGGE profiles of the amplicons of caecal samples from wild (WR, n ¼ 12) and farm rabbits given diets with high (HSF, n ¼ 4) or low (LSF, n ¼ 4) content of soluble fibre.
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was also lowest in WR (P < 0.001), but in this case no differences due to the diet ingested in farm animals were detected. On average, 10.5 bands were detected from the DGGE analysis of Archaea community in WR, whereas among farm rabbits LSF showed more bands than HSF (21.8 and 11.3, respectively). Consequently, Evenness index were highest for LSF, and lowest for WR (P < 0.001), whereas Shannon index did not shown differences between HSF and WR, thus indicating a higher biodiversity in the Archaea community in farm rabbits given the former diet. 4. Discussion
Fig. 4. Dendrogram obtained from the DGGE analysis of the Archaeal community of caecal samples from wild (WR) and farm rabbits given diets with high (HSF) or low (LSF) content of soluble fibre. The scale shows the proportion of similarity.
different bands were detected in the gel, but whereas the average number in LSF was 21.8, it only reached 11.3 and 10.5 in HSF and WR, respectively. As it was observed for bacterial diversity, farm rabbits clustered together depending on their diet, reaching similarities of 0.50 and 0.53 for LSF and HSF, respectively. All wild rabbits were grouped in a different cluster, showing similarities below 0.40 with both groups of farm animals. Within-group diversity was higher in WR, and barrow did not show any apparent effect. Total concentration (ng DNA per mg fresh content) and microbial biodiversity estimated by the Shannon and Evenness indexes, for bacterial and archaeal communities in the rabbit caecum are shown in Table 2. Differences were detected in total concentration of bacteria (P < 0.001), being highest in farm rabbits given HSF, intermediate in LSF and lowest in WR. Average number of bands detected for LSF and HSF bacterial communities were 28.3 and 29.3, and there were no differences in biodiversity between farm rabbits despite the diet they fed. However, wild rabbits (18.4 bands) showed a lower diversity (P < 0.01). Archaeal total concentration
Table 2 Total concentration (ng DNA/mg fresh content) and Shannon and evenness indexes in bacterial and Archaea communities in the caecum of wild rabbits (WR, n ¼ 16 for bacteria and n ¼ 12 for Archaea) and farm rabbits given low (LS, n ¼ 4) and high (HS, n ¼ 4) levels of soluble fibre.
Bacteria Total concentration Shannon index Evenness index Archaea Total concentration Shannon index Evenness index
WR
LSF
HSF
SDa
Probability
15.719c 2.848b 0.676b
46.166b 3.339a 0.776a
71.441a 3.368a 0.830a
9.9679 0.3123 0.0744
<0.001 0.005 0.005
2.923b 2.332b 0.582c
13.394a 3.071a 0.792a
13.524a 2.397b 0.697b
4.4856 0.2082 0.0506
<0.001 <0.001 <0.001
Within rows, different letters indicate significant differences among treatment means (P < 0.05). a SD, standard deviation.
DGGE profiling was performed in order to obtain insight into the complexity of the microbial community of rabbit caecum, and to compare the community inhabiting fermentation sites of hosts from two different environments and feeding conditions, farm vs. wild rabbits. The presence of protozoa in caecal samples was not detected in any rabbit, and therefore no results are presented here. Previous studies in our laboratory [2] also failed to detect protozoa in caecal contents of rabbits, either by optical or molecular approaches. To our knowledge, there is no published report of the presence of protozoa in this host species. Whereas the protozoa might interact with archaeal community in the rumen and they are present in some hindgut fermentors such as the horse [29], this does not seem to be the case in the rabbit caecum. Feeding habits in wild rabbits are greatly conditioned by the distance to barrow, in order to prevent for predation [30], although this also depends on other factors such as feed abundance, predatory pressure or population density. Grasses high in structural polysaccharides are their main feed source [4] but, when feed is scarcely available, as it should be the case in this experiment (winter), rabbits tend to select the most nutritive parts of plants [4,5]. In any case, because of the season when the experiment was carried out the diet of wild rabbits should probably include high proportions of both NDF and soluble fibre, as well as a very low proportion of starch, in comparison with farm rabbits. Caecal environmental characteristics (Table 1) did not differ between farm rabbits, despite of their differences in soluble fibre contents. Both ammonia and total VFA concentration were in the upper range of previous observations [2,31], probably because of their high level of both soluble and insoluble fibre. In wild rabbits, caecal pH was more than 0.45 units lower than in farm ones, as a consequence of their high concentration of VFA that was 0.39 higher than that of farm animals. This should imply either a higher input of fermentable substrates reaching the caecum or an increased retention time of them into the organ. Besides, the VFA profile is characterised by a lower acetate and larger butyrate proportion in wild compared with farm rabbits. The capability of WR during winter to select vegetal parts rich in soluble sugars, that promote a higher butyrate concentration, rather than cell wall polysaccharides, that would increase the contribution of acetate, should help to explain these responses. The lower ammonia concentration in the caecum of WR could be associated to a lower protein input or a reduced proteolytic activity, since the lower microbial concentration in these animals discards a higher nitrogen utilisation by microbes. The lack of information about the feeding preferences of wild rabbits in terms of protein intake makes this question difficult to solve. The absence of differences in VFA caecal concentration between LSF and HSF despite the higher proportion of rapidly fermentable compounds in the latter is probably caused by an enhanced utilisation of available energy towards bacterial synthesis, which in fact is the major goal of caecum in rabbits [2], instead of fermentation itself. This also agrees with the lack of differences in abundance of Archaea between LSF and HSF, since
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the hydrogen required for methanogenesis comes from production of acetate and butyrate that did not differ among these treatments (Table 1). The fact that wild rabbits fed diets with high NDF [4,6] compared with farm rabbits partly explains the differences in total bacterial concentration in the caecum (Table 2), since the slow fermentability of NDF would contribute to impairing the rate of microbial protein synthesis [32]. Wallage-Drees and Deinum [4] suggest that, during winter time, wild rabbits may suffer an important shortage of energy intake, being even below the standards for maintenance. Besides, this low microbial concentration could also be related to a low protein input to the caecum in wild rabbits during the winter season, although the lack of information about their diet in this work does not allow supporting this thought. In contrast, differences in bacterial concentration among farm animals would depend on the level of soluble fibre, since a high input of highly digestible matter to the caecum in HSF increases bacterial growth [14,33]. Average proportion of DNA from Archaea in the caecal contents ranged from 0.16 to 0.22 of total microbial DNA, which is within the range observed by [9]. The presence of Archaea was detected in the caecum of all wild and farm rabbits. Belenguer et al. [31] observed a high variability in methane production in vivo, with only 2 out of 16 farm rabbits showing a significant concentration; however, these authors detected methane in all cases when caecal contents of such animals were incubated in vitro, thus indicating the presence of Archaea in all animals, even at low concentrations sometimes. It seems that methanogenic microorganisms in the large intestine must compete for H2 with others such as acetogenic and sulphate-reducing bacteria [34], the prevalence of one of these microbial groups probably depending on the rate of passage [35]. Further, methanogenic microorganisms are especially pH sensitive [36], and this parameter in WR was 0.45 units lower than in farm rabbits, which might explain the lower archaeal concentration in WR. In any case, the proportion of Archaea in WR compared to farm rabbits (0.22) was similar to that of bacteria (0.27). As it was reported recently for the rumen environment, the size of the methanogen population does not necessarily correlate with differences in feed efficiency, diet, or metabolic measurements [37]. Thus, the structure of the methanogenic community at the genus or species level may be more important for determining host feed efficiency under different dietary conditions [38]. The number of bands in the DGGE gel for Archaea detected in our study was 10e11 for both WR and HSF (Fig. 3), with is in the line of the scarce diversity of Archaea detected by [17] who reported lower methanogen diversity in the rabbit caecum compared with the fermentation compartments of ruminants and pigs. However, when rabbit caecum samples from this experiment were compared to others from rumen or faeces from goats [39], it was observed that rabbits hold a microbial community of similar complexity than that observed in ruminants. This fact might be due to the specificity of the set of primers used, 519f/915rGC, that was reported to be more suitable for studying the methanogenic archaeal community in the rumen in a comparison of nine primers set. Kusar and Avgustin [17] also indicated that the Archaea community of the rabbit caecum is unique and different to that of the rumen or pig faeces. They stated that one species of the genus Methanobrevibacter may dominate, as it has also been suggested by [40] in chickens. However, the number of bands was almost two-fold (22 bands) when farm rabbits were given LSF. This is difficult to explain, since NDF content was the same in both LSF and HSF diets, and fermentation parameters were almost identical (Table 1) but only two bands were present in all samples. In any case [41], suggest that there is large diversity in the methanogenic digestive community among individuals of the same species, depending on their location and feeding conditions.
The reported methanogenic PCR-DGGE profiles that have been detected in the rumen are greatly affected by diet [38]. Between low- and high-energy diets, the major pattern changed from a community containing predominantly Methanobrevibacter ruminantium NT7 in the first case to another where Methanobrevibacter smithii, Methanobrevibacter sp. AbM4, and/or M. ruminantium NT7 predominates in the latter. For each diet, the methanogenic PCRDGGE pattern was strongly associated with the host feed efficiency. 5. Conclusions Caecal fermentation characteristics differ between wild and farm rabbits, despite the level and kind of dietary fibre, thus highlighting differences in dietary habits. In a similar sense, these types of rabbits harbour clearly different bacterial and archaeal communities. It is worth noting that, despite the intra-group similarity, the number of archaeal species in the caecum of wild rabbits was close to that of farm rabbits given high-soluble fibre diets, which may indicate a selective feeding in the former. In farm rabbits, diversity is influenced by the dietary level of soluble fibre. To our knowledge, this is the first approach to the study of digestive ecosystem of wild rabbits, and its comparison with farm rabbits might directly explain the effect of environment and dietary habits. Acknowledgements This work was financed through the Projects AGL 2006-07596 from the Ministerio de Educación y Ciencia (Spain). The stage of Ms. Norelys Rodríguez-Romero in the University of Zaragoza was financed though a Doctoral fellowship from the Universidad Nacional Experimental del Táchira (Venezuela). Dr. L. Abecia acknowledges the receipt of a research contract from the Spanish National Research Council (CSIC, JAE-Doc Programme). References [1] MARM. Anuario de Estadística 2010. Medio natural: caza y pesca fluvial (cap. 12.5). Medio Rural y Marino, Gobierno de España: Ministerio de Agricultura, http://www.marm.es/estadistica/pags/anuario/2010; 2011 [last accessed 15/12/2011]. [2] Belenguer A, Balcells J, Fondevila M, Torre C. Caecotrophes intake in growing rabbits estimated either from urinary excretion of purine derivatives or from direct measurement using animals provided with a neck collar: effect of type and level of dietary carbohydrate. Anim Sci 2002;74:135e44. [3] Gidenne T, Pérez JM. Effect of dietary starch origin on digestion in the rabbit. 2. Starch hydrolysis in the small intestine, cell wall degradation and rate of passage measurements. Anim Feed Sci Technol 1993;42:249e57. [4] Wallage-Drees JM, Deinum B. Quality of the diet selected by wild rabbits (Oryctolagus cuniculus L.) in autumn and winter. Neth J Zool 1986;36:438e48. [5] Martins H, Milne JA, Rego F. Seasonal and spatial variation in the diet of the wild rabbit (Oryctolagus cuniculus) in Portugal. J Zool. London 2002;258: 395e404. [6] Soriguer RC. Alimentación del conejo (Oryctolagus cuniculus L.) en Doñana, SO España. Doñana, Acta Vertebrata 1988;15:141e50. [7] Boulahrouf A, Fonty G, Gouet P. Establishment, counts and identification of the fibrolytic bacteria in the digestive tract of rabbit. Influence of feed cellulose content. Curr Microbiol 1991;22:1e25. [8] Padilha MTS, Licois D, Gidenne T, Carre B, Fonty G. Relationships between microflora and cecal fermentation in rabbits before and after weaning. Reprod Nutr Develop 1995;35:375e86. [9] Bennegadi N, Fonty G, Millet L, Gidenne T, Licois D. Effects of age and dietary fibre level on caecal microbial communities of conventional and specific pathogen-free rabbits. Microb Ecol Health Dis 2003;15:23e32. [10] Sirotek K, Marounek M, Rada V, Benda V. Isolation and characterization of rabbit caecal pectinolytic bacteria. Folia Microbiol 2001;46:79e82. [11] Abecia L, Fondevila M, Balcells J, Edwards JE, Newbold CJ, McEwan NR. Molecular profiling of bacterial species in the rabbit caecum. FEMS Microbiol Lett 2005;244:111e5. [12] Monteils V, Cauquil L, Combes S, Godon JJ, Gidenne T. Potential core species and satellite species in the bacterial community within the rabbit caecum. FEMS Microbiol Ecol 2008;66:620e9.
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