COBISS: 1.08 Agris category code: L51 SINGLE AND COMBINED EFFECT OF DIETARY THYME (Thymus vulgaris) AND SPIRULINA (Arthrospiraplatensis) ON BACTERIAL COMMuNITY IN THE CAECuM AND CAECAL FERMENTATION OF RABBITS Viola VÄNTUS Andras BONAI *, Attila ZSOLNAI *, Alessandro DAL BOSCO 2, Zsolt SZENDRÖ Gabor TORNYOS Zsofia BODNÄR *, Wael Awad MORSY 3, Roland POSA *, Maria TOLDI *, Brigitta BOTA *, Melinda KOVÄCS Antonella DALLE ZOTTE 4 ABSTRACT The objective of this study was to evaluate the effect of supplementation of the growing rabbits' diet by Spirulina or/and Thyme (food supplement) on the composition and amount of the microbiota and production of volatile fatty acids of the rabbits' caecum. We used classical culturing methods and after bacterial DNA extraction, the quantity of bacteria (belonging to phylum Firmicutes and Bacteroidetes) were determined by qPCR reactions with the aid of bacterial ribosome coding DNA at the Molecular Biology Laboratory, University of Kaposvar. The experiment was carried out using rabbits (Pannon White) from rabbit-farm of Kaposvar University. Young rabbits were weaned on the 35th day after birth. All experimental animals were the same age. Duration of Spirulina and/or Thyme supplementation -after weaning- was 48 days. Samples were collected on the 14th, 28th and 48th day of the supplementation. No dietary effect on pH of the caecal content was detected. The number of Escherichia coli, total anaerobic and strictly anaerobic bacteria decreased by age, no effect of the diet could be demonstrated. Supplementation with Thyme resulted in slightly higher ratio of propionic acid, but the difference was not significant. In conclusion, Spirulina and/or Thyme supplementation of diet after weaning had no substantial effect on the volatile fatty acid (VFA) production, while the classical microbiological determination did not find a substantial effect on the composition of the caecal microbiota. By the Quantitative PCR method we measured significantly lower bacterial copy numbers in the samples of ST treated group. This indicates that the ST (Spirulina 5% and 3% Thyme) feed supplementation has an antimicrobial effect on the investigated bacterial groups in the caecum of growing rabbit. Key words: rabbits / animal nutrition / Spirulina / Thyme / microbiology/ caecal microbiota / volatile fatty acids 1 INTRODUCTION The composition and the activity of the caecal microbiota have a strong influence on health, because of their role in nutrition, pathogenesis and immune function (Gibson and Roberfroid, 1995). In addition to classical culturing methods, molecular microbiology techniques have been recently introduced for determination of changes in the microbiota. In connection with the ban of using antibiotics as growth promoters in the EU several studies have been carried out on different feed additives - for example herbal extracts - as alternatives for antibi- otics, because of their growth-promoting effects. Thyme has been reported for its antimicrobial and antioxidant properties (Dorman and Deans, 2000). Spirulina is a type of blue-green algae that is rich in protein, vitamins, minerals, and carotenoids. It has been used as human food supplement for over 20 years, because of its high nutrient content, including B complex vitamins, beta-carotene, vitamin E, manganese, zinc, copper, iron, selenium, and gamma linolenic acid (Belay et al., 1996). Several studies have been shown that Spirulina has beneficial biological activities, such as immunomodulation, antioxidant, anticancer, antimicrobial and probiotic effects (Belay, 2002). 1 Kaposvar Univ., Fac, of Animal Science, Guba S. 40, 7400 Kaposvar, Hungary, e-mail: kovacs.melinda@ke.hu 2 Dept. of Applied Biology, Univ. of Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy 3 Animal Production Research Institute, Agricultural Research Center, Ministry of Agriculture, Dokki, Cairo, Egypt 4 Dept. of Animal Medicine, Production and Health, Univ. of Padova, Agripolis, Viale dell'Università 16, 35020 Legnaro (PD), Italy The objective of this study was to evaluate the effect of supplementation (between the ages of 5-11 weeks) of the growing rabbits' diet by Thyme and/or Spirulina on the bacterial community and fermentation in the rabbits' caecum. 2 MATERIALS AND METHODS 2.1 ANIMALS AND EXPERIMENTAL DESIGN Rabbits involved in the experiment received the control pellet (C) from the age of 3 weeks. After weaning (at the age of 5 weeks) the rabbits were housed in wire net cages (0.61 x 0.32 m, 3 rabbits/cage). The temperature and daily lighting in the house were 16-18 °C and 16 hours, respectively. The weaned rabbits were randomly sorted to 4 groups (42 rabbits/group). Rabbits of the control group (C) received a pellet without any supplementation throughout the experiment (up to11 weeks of age). In the other groups the pellet was completed by 5% Spirulina (S), or 3% Thyme (T) or by both (ST) for the whole (5-11 week) growing period. More details about housing and feeding conditions are described in Gerencser et al. (2012). 2.2 SAMPLE COLLECTION During the treatment at 14, 28 and 48 days (sampling points: 1, 2, 3, respectively) 6 healthy animals from each group (one animal/cage) were randomly selected and slaughtered at 02:00 p.m. The digestive tract was removed immediately and the caecum was separated. The quantity of the fresh caecal content was measured and pH value was determined. For microbiological determination, 1 g of the fresh caecal content was homogenised at room temperature and serial dilution was made with sterile isotonic solution under a carbon dioxide stream. For molecular tools and chemical analysis, some of the fresh caecal contents was frozen and stored at -20 or -80 °C until processing. 2.3 MICROBIOLOGICAL DETERMINATION Total aerobic bacteria and total anaerobic bacteria were cultured on blood agar, prepared with 5% defibri-nated calf blood. Samples were incubated at 37 °C for 24 h and 72 h, under aerobic and anaerobic condition, respectively. Coliforms and Escherichia coli sub-spp. were cultured on a Chromocult differentiation medium (Merck, Darmstadt, Germany). Samples were incubated at 37 °C, under aerobic conditions, for 24 h. Campylobacter spp. was cultured on blood-free selective agar (Merck). Samples were incubated at 37 °C, under aerobic conditions, for 48 h. The obligate anaerobe organisms were cultured on Schaedler's agar (Scharlan Chemie, Barcelona, Spain), the selectivity of which was increased by the addition of esculin, neomycin (Merck) and Ferri-ammonium-citrate (Scharlan Chemie, Barcelona, Spain). Petri dishes were placed into Anaerocult culture dishes (Merck), in which the anaerobic conditions were ensured with the help of an Anaerocult A (Merck) gasifying bag. The samples were incubated 37 °C for 96 h. After the incubation had elapsed, the colonies were counted (ISO 4833:2003) with Acolyte colony counter (Aqua-Terra Lab, Veszprem, Hungary). The colony counts were expressed in log10 colony forming units (CFU) related to 1 g of sample. 2.4 DETERMINATION OF VOLATILE FATTY ACIDS (VFA) CONCENTRATION VFA concentrations of 11 week old growing rabbits' caecal samples were measured. Approximately 5 g digesta per rabbit were stored in fridge (-20 °C) until measurement. The concentration of VFA was measured by gas chromatography (Shimadzu GC 2010, Japan), using external standard. 2.5 DNA EXTRACTION AND QPCR Total DNA from about 200 mg of caecal sample of ST treated animals was extracted and purified using the QIAamp ® DNA Stool Mini Kit (50) (QIAGEN) according to the manufacturer's instructions. DNA concentrations were measured using Smart Spec Plus Spectrophotom-eter (BioRad). The concentrations of all DNA samples were set to 60 ng/^l. After the preparation of caecal samples (bacterial DNA extraction) the quantity of bacteria (belonging to phylum Firmicutes and Bacteroidetes) were determined by QPCR reactions. QPCR was carried out in a 25 |l/tube reaction mixture containing 12.5 |l Brillant II SYBR QPCR Low Rox Master Mix (Agilent Technologies), 0.2 |M of each primer (Table 1.), 10.5 |l sterile distilled water and 1 |l of DNA extract (60 ng/^l). Sample measuring robot (QIAgility, QIAGEN) was used to fill 96-well plates with PCR reaction components and samples, which - compared to manual measurement -reduced the standard deviations. The PCR program consisted of 10 min at 95 °C, 40 cycles with 30 sec at 95 °C, 1 min at 60 °C. MxPro 3000P QPCR apparatus (Agilent Technologies) was used for the bacterial target sequence ampli- Table 1: Oligonucleotide sequences used for QPCR Group Item Oligonucleotide sequence (5'-3' ) Ta * (°C) References Clostridium Forward (Cc1) GAC GCC GCG TGA AGG A 60 Firmesse et al. (2008) coccoides Reverse (Cc2) AGC CCC AGC CTT TCA CAT C Clostridium Forward (Cl9) CCT TCC GTG CCG SAG TTA 60 Firmesse et al. (2008) leptum Reverse (Cl8) GAA TTA AAC CAC ATA CTC CAC TGC TT Bacteroides Forward (Bs2) CCT WCG ATG GAT AGG GGT T 60 Firmesse et al. (2008) Reverse (Bs1) CAC GCT ACT TGG CTG GTT CAG *T - Annealing temperature fication applying primers and SYBR Green in the experimental assembly. Specificity of PCR reactions were checked by melting point analysis. All samples were measured in three technical triplicates. Ct values of the samples - having equilibrated concentrations - were the basis of monitoring the changes of bacterial community. After cloning of the amplified PCR products (external lab orders), we determined the plasmid concentrations, and dilution series were prepared (standard curve). The bacterial contents of samples were calculated with the aid of that. The obtained copy numbers of the samples were adjusted to one gram of caecal content. The bacterial quantification based on real-time PCR. Real-time PCR has been developed for monitoring the amplification reaction. SYBR Green I is a double-stranded DNA binding dye that allows the detection of PCR products, including the DNA extracted from bacterial samples. The bacterial quantification data using realtime PCR is commonly expressed as absolute quantities in units such as copies/g, colony-forming unit (CFU)/mL or Log CFU/g of samples such as digesta. In Quantita- tive PCR/qPCR gives realtime monitoring, each cycle is detected via light (monochromatic or narrow wavelength range) exitation of a dsDNA binding fluorescent dye (SYBR Green): the intensity of the emitted light correlates with the amount of product generated by PCR, allowing quantitative detection (Navidshad et al. 2012). 2.6 DATA ANALYSIS Data from classical microbiological determination and gas chromatography were analysed by using GLM (General Linear Model) procedure of SPSS (2002), version 10.0. Group of diet and age were factors. The formula of General Linear Model included the following: y.j = ^ + sampling point. + diet. + sampling point, x diet + e.. j i where y is the measured pH values, etc. (see Table 2., column 1), ^ is the general mean, sampling point is the Table 2: Effect of different pellets inclusion on caecal digesta traits of growing rabbits Traits Experimental diets Sampling point (d) P-value C S T ST 1 2 3 Sampling N 18 18 18 18 24 24 24 RSD Diet point pH of caecal 6.37 6.46 6.44 6.49 6.35 6.50 6.47 0.23 0.501 0.122 cont. Total aerobic 6.91 6.87 7.02 6.53 7.02 6.91 6.57 0.71 0.128 0.055 bacteria 1 E. coli spp. 1 3.95 3.92 3.70 3.50 3.91b 4.19b 3.21a 0.84 0.225 0.000 Campylobacter 3.81 4.01 3.59 3.51 3.80 3.66 3.01 0.86 0.536 0.577 spp. 1 Total anaerobic 8.61 8.52 8.54 8.39 8.82b 8.31a 8.39a 0.43 0.357 0.000 bacteria1 Strictly anaero- 7.95 7.86 8.00 7.92 8.05b 8.00b 7.75a 0.35 0.642 0.005 0.830 0.112 0.093 0.457 bic bacteria1 1 Germ counts expressed in loglO CFU/g caecal digesta. Different superscripts mean significant differences between ages. Table 3: Effect of different pellets inclusion on VFA content of the caecal digesta at 77 days of age, n = 6 Experimental diets Traits C Sd S sd T sd ST sd P-value Dry matter content (w/w%) 22.6 1.12 22.5 1.60 22.6 0.39 22.5 0.87 0.990 Total VFA content (mmol/kg) 35.1 5.40 31.0 9.58 34.3 6.40 36.1 10.0 0.715 Acetic acid %1 74.8 1.78 73.6 1.60 74.1 1.12 73.9 1.17 0.516 Propionic acid %1 6.97 0.53 7.19 1.26 8.46 1.33 7.83 0.93 0.095 Butyric acid %1 18.2 1.75 19.3 2.17 18.8 4.29 17.3 2.12 0.659 1 Proportion within total VFA content event of caecal content sampling (1, 2, 3), indicates the impact of the combined supplemented diet and e is the residual error. VFA content of 11th week old rabbits' caecal digesta was analysed by using one way ANOVA. The significance of differences was tested by Tukey post hoc test. The copy numbers of the investigated bacterial groups were visualised in copy numbers vs. samplingevents coordinates (data not shown). The qPCR dataset was analyzed with the GLM to determine differences between the amount of investigated bacterial groups, where the sampling points and ST (Spirulina 5% and Thyme 3%) supplemented diet were included as fixed effects and bacterial copy numbers as dependent variable. The formula of General Linear Model included the following: y.. = ^ + sampling point. + diet. + sampling point, x diet + e.. J i where y is the copy number of the investigated bacteria (e.g. Bacteroides), ^ is the general mean, sampling point is the event of caecal content sampling (1, 2, 3), indicates the impact of the combined supplemented diet and e is the residual error. The significance of differences was tested by Tukey post hoc test. 3 RESULTS AND DISCUSSION No effect of the diet on the pH of caecal content was detected (Table 2). The number of E. coli, total anaerobic and strictly anaerobic bacteria decreased by age, and no effect of the diet could be demonstrated. Supplementation with Thyme resulted in a slightly higher ratio of propionic acid, but the difference was not significant (Table 3). When using the molecular genetics tool, qPCR, all investigated bacterial group displayed the similar, increasing trend in the control and ST treated group (Table 4.). The amount of Bacteroides, Clostridium leptum and Clostridium coccoides in 1 g caecal content of the ST treated samples were significantly less (P < 0.05) (Table 5.) than that of the control group at each sampling point. However the classical microbiological determination was able to demonstrate significant differences between the sampling points in case of E. coli, total anaerobic and strictly anaerobic bacteria (suggesting declined bacterial load with age, Table 2.), this significance clearly disappears when the effect of the investigated diet -as an interaction with sampling points- was included into the calculations. As a consequence we cannot state any substantial effect of supplementing the diet by Spirulina and/ or Thyme, after weaning on the composition of the caecal microbiota based on classical microbiological approach. By Quantitative PCR method we measured increasing copy numbers of bacteria with age both in the con- Table 4: Average copy number of 1 gram caecum samples at different sampling points and their deviations Sampling point -day of the treatment Number of copies Bacteroides Clostridium leptum Clostridium coccoides C D ST D C D ST D C D ST D 1 (14) 97034 2022 91530 910 95707 2082 89717 831 110437 2609 102985 1011 2 (28) 97052 2539 97683 2361 96974 5399 95603 2487 109212 7812 109837 2739 3 (48) 99275 504 99028 1715 99018 2895 96673 1707 114646 3494 111055 2008 C - Control, D - Deviation, ST - Spirulina and Thymus combined diet 80 Acta agriculturae Slovenica, Supplement 3 - 2012 Table 5: Differences between the Control (C) and treated (ST) groups, results of the Tukey post-hoc test Mean Standard diff. error P-value Bacteroides C-ST 2596 718 0.005 Clostridium coccoides C-ST 4950 1435 0.007 Clostridium leptum C-ST 3663 1229 0.020 trol (C) and in the SP diet (Spirulina 5% and 3% Thyme) diet. Comparing copy number values of SP diet to that of the corresponding control groups, antimicrobial effects (P < 0.05) were demonstrated on the investigated bacterial groups in the caecum. The reason of this difference from the classical microbiological approach may come from the sensitivity of molecular microbiology techniques (Tannock et al., 2000; Takahiro et al., 2003). In case of Firmicutes (Clostridium leptum, Clostridium coccoides) these observed trends are in good accordance to the findings in humans and rabbit, such as body weight (Turnbaugh et al., 2009) and age (Combes et al., 2011) and are associated with a larger proportion of Firmicutes. In the present study, the dietary change modifies the investigated bacterial groups, but the quantity of total bacteria has not been measured by qPCR. The decrease of proportion of some species as a consequence of a change in substrate availability could be compensated for by the increase of proportion of other groups better adapted to the available nutrients (Zoetendal et al., 2004). The consumption of resources that govern ecosystem processes through two types of effects: a 'complementarity effect', which occurs through either resource partitioning or fa-cilitative interactions between species and a 'selection effect', which occurs whenever species diversity is correlated with the chance of resource use (Cardinale et al., 2002).The trend is valid for each test group, not the total number of bacteria. 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