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British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Effect of thyme essential oil and selenium on intestine integrity and antioxidant status of broilers a
a
a
a
a
a
I. Placha , J. Takacova , M. Ryzner , K. Cobanova , A. Laukova , V. Strompfova , K. b
Venglovska & S. Faix a
a
Institute of Animal Physiology, SAS, Kosice, Slovak Republic
b
Faculty of Science, Institute of Biology and Ecology, Pavol Jozef Safarik University, Kosice, Slovak Republic Accepted author version posted online: 08 Jan 2014.Published online: 16 Apr 2014.
To cite this article: I. Placha, J. Takacova, M. Ryzner, K. Cobanova, A. Laukova, V. Strompfova, K. Venglovska & S. Faix (2014) Effect of thyme essential oil and selenium on intestine integrity and antioxidant status of broilers, British Poultry Science, 55:1, 105-114, DOI: 10.1080/00071668.2013.873772 To link to this article: http://dx.doi.org/10.1080/00071668.2013.873772
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
British Poultry Science, 2014 Vol. 55, No. 1, 105–114, http://dx.doi.org/10.1080/00071668.2013.873772
Effect of thyme essential oil and selenium on intestine integrity and antioxidant status of broilers I. PLACHA, J. TAKACOVA, M. RYZNER, K. COBANOVA, A. LAUKOVA, V. STROMPFOVA, K. VENGLOVSKA1 AND S. FAIX
Downloaded by [UQ Library] at 10:57 16 June 2014
Institute of Animal Physiology, SAS, Kosice, Slovak Republic, and 1Faculty of Science, Institute of Biology and Ecology, Pavol Jozef Safarik University, Kosice, Slovak Republic
Abstract 1. This study evaluated the duodenal wall integrity, antioxidant status as well as some immunological parameters of broiler chickens supplemented with 0.5 g Thymus vulgaris essential oil (EO)/kg diet and 0.4 mg Se/kg DM (dry matter) derived from sodium selenite. 2. A total of 192 one-d-old randomly divided chickens of both sexes (Ross 308 hybrid broilers) were divided into 4 treatment groups of 48 birds each. 3. The first group was fed on a nutritionally balanced basal diet (BD). The other three groups received BD supplemented with 0.5 g/kg thyme oil, or 0.4 mg Se/kg DM, or both feed additives together. 4. The results for the evaluated feed additives were (1) thyme oil – decreased malondialdehyde (MDA) concentration in duodenal mucosa and kidney, increased immunoglobulin A (IgA) concentration in duodenal mucosa, stimulated phagocytic activity in blood, improved intestinal barrier integrity (2) selenium – increased glutathione peroxidase (GPx) activity in blood and liver as well as thioredoxin reductase (TrxR) activity in duodenal mucosa, liver and in the kidney, (3) EO with selenium – increased thioredoxin reductase (TrxR) activity in duodenal mucosa. 5. These results demonstrated that thyme oil alone showed more effective potential to improve intestinal barrier integrity and antioxidant status as well as evoking an immune response in chickens, than if diets were supplemented with both thyme oil and selenium.
INTRODUCTION Since antibiotics, as feed additives, were banned in the European Union in 2006, research into alternatives has gained in importance. Besides probiotic microbiota and synthetic antioxidants, phytogenic substances are most commonly used for the same objective. The market for plant-based performance enhancers has increased since the 1990s. For example, sales of essential oils (EOs) in the EU amounted to 90 t in 1996, while only 10 years later, they reached 600 t (Greathead, 2003). Synthetic antioxidants are currently approved for controlling lipid oxidation in foods, but there is consumer concern over their use (Botterweck et al., 2000). Much attention is now given to the
application of natural antioxidants for stabilising foods against oxidation (Lim et al., 2001). Gut health is a major topic for research, not only in humans but also in animals. Anything that affects the health of the gut will undoubtedly influence the animal as a whole, and consequently alter its nutrient uptake and requirements (Choct, 2009). Hydrogen peroxide is constantly generated within all cell types, including gastric epithelial cells, and has been implicated as a mediator of gastrointestinal injury. Gastric epithelial monolayers, which are exposed to reactive oxygen species (ROS), may cause a significant decrease in trans-epithelial electrical resistance (TEER), which means the transport of ions across the epithelium. When this transport is restricted, the
Correspondence to: I. Placha, Institute of Animal Physiology, Slovak Academy of Sciences, Soltesova 4, 04 01 Kosice, Slovak Republic. E-mail: [email protected] Accepted for publication 18 October 2013.
© 2014 British Poultry Science Ltd
Downloaded by [UQ Library] at 10:57 16 June 2014
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electrical potential gradient increases across the epithelia. The intestinal barrier is important in the pathogenesis of various diseases. To elucidate passage routes and mechanisms involved in intestinal absorption and permeation, a widely used tool is the Ussing chamber, which was first described in 1951 (Ussing and Zerhan, 1951). EOs are able to scavenge some free radicals, thus playing an important role in prevention of some diseases such as brain dysfunction, cancer, heart disease and immune system decline. Increasing evidence suggests that these diseases may result from cellular damage caused by free radicals. In addition to their ability to scavenge free radicals, there is also evidence that some EOs develop anti-inflammatory activity. For example, chamomile EO has been used for centuries because of its anti-inflammatory properties (Kamatou and Viljoen, 2010). The anti-inflammatory activity of EOs may be attributed not only to their antioxidant activities, but also to their interaction with signalling cascades involving cytokines and regulatory transcription factors, and on the expression of proinflammatory genes (Miguel, 2010). We found in our previous experiment that thyme oil at a concentration of 0.5 g/kg DM (dry matter) can sufficiently control the release of ROS from neutrophils, and by this way, it could simultaneously stimulate neutrophils thus improving phagocytic activity (Placha et al., 2013). Selenium is an essential micro-mineral element with numerous functions such as regulating metabolism, improving immunity, enhancing reproductive performance, preventing cancer and resisting free radicals (Yang et al., 2009). This essential nutrient has a special function in antioxidant control mechanisms as an essential component of the active centre of selenoenzymes (Zuberbuehler et al., 2006). The routine source of Se used to supplement animal feeds is sodium selenite. However, this inorganic source of selenium has been shown to provide relatively low selenium retention (Boldižárová et al., 2005). Even though organic selenium is a superior source because of its active absorption from the intestine in comparison with passive diffusion of sodium selenite from the intestinal tract, inorganic sodium selenite is still used as the principal source of selenium in animal feeds (Mahan, 1995; Schrauzer, 2001; Edens et al., 2002). Brenes and Roura (2010) stated that synergistic effects between EOs and other feed additives could be exploited for maximisation of their antioxidant properties, antibacterial activity and ability to stimulate the immune system. These interactions need to be investigated. The antioxidant activities and role of EOs as well as organic and inorganic forms of selenium in the immune system of chickens are well documented, but their synergism or antagonism has
received little interest to date. Therefore, the aim of our study was to examine whether the administration of Thymus vulgaris EO and selenium alone or together would have a stronger effect on intestinal integrity, parameters of antioxidant status as well as on some immunological parameters in broiler chickens. In addition, their influence on selected microbiota was investigated.
MATERIALS AND METHODS Animal care and use The experiment was carried out in accordance with the established standards for use of animals. The protocol was approved by the Ethical Commission of the Institute of Animal Physiology, Slovak Academy of Sciences in Košice, Slovakia and by Slovak governmental authority (Č.k. RO-820/10-221).
Experimental design and housing A total of 192 Ross 308 hybrid broilers of both sexes was randomly divided at the day of hatching into 4 dietary treatments of 48 birds each. Each dietary treatment consisted of 6 replicates. A replicate was a cage with 8 birds. All cages were placed in the same room, in which temperature was controlled during the experiment. Chickens were reared with a lighting regimen of 23 h of light and 1 h of darkness. The initial room temperature of 32–33°C was reduced weekly by 3°C to a final temperature of 23°C. All birds had free access to water and feed. Animals were fed for 5 weeks on the 4 experimental diets. The first group was given the basal diet (BD, BIOFED – HYD-02, Kolárovo, Slovakia, Table 1); the second was fed on the same BD enriched with 0.5 g/kg of T. vulgaris EO. The third group received BD supplemented with 0.4 mg Se/kg DM derived from sodium selenite (SS), and the 4th was fed on BD enriched with both EO and SS together. The mean analysed values of the Se content in the diets [starter (0–7 d), grower (8–21 d) and finisher (22– 35 d)] for groups 1, 2, 3 and 4 were 0.21 ± 0.01, 0.20 ± 0.03, 0.56 ± 0.05 and 0.60 ± 0.07 mg/kg DM, respectively. At the age of 5 weeks, 24 chickens from each treatment (4 animals/replicate) in the best physical and health condition were sacrificed. Samples of liver, kidney and duodenal mucosa tissues were collected for biochemical analysis and stored at – 70°C until analysis. Their duodenum was separated to measure TEER in vitro. Blood samples for analyses were collected into heparinised tubes.
DIETARY THYME AND SELENIUM
Table 1.
107
Ingredients and chemical composition of the basal broiler diet
Ingredients
g/kg
Composition
g/kg
Wheat, ground Maize, ground Soya bean meal, extracted Fish meal Blood meal Limestone Monocalcium phosphate Sodium chloride Premix 1 Sunflower oil
340 400 180 20 10 10 8 3 19 10
Dry matter Crude protein Ash Crude fibre Calcium Phosphorus Lysine Methionine Methionine + cystine Linoleic acid Calculated MEn (N-adjusted metabolisable energy, MJ/kg)
891 190 50 35 7.0 5.0 9.5 4.0 7.5 10.0 12.0
Crude protein, dry matter and selenium are analysed data. The analysed selenium content of the BD was 0.2 mg/kg DM. 1The vitamin/mineral premix provided per kg of complete diet: retinyl acetate: 3.6 mg; cholecalciferol: 0.1 mg; menadione: 4 mg; tocopherol: 100 mg; thiamine: 3 mg; riboflavin: 9 mg; niacin: 60 mg; pantothenic acid: 15 mg; pyridoxine: 6 mg; cyanocobalamin: 0.04 mg; biotin: 0.2 mg; folic acid: 2 mg; lysine: 9.5 g; methionine: 4.0 g; zinc: 100 mg; iodine: 1 mg; cobalamin: 0.4 mg; manganese: 110 mg; copper: 15 mg; iron: 120 mg.
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Thyme oil used in the model experiment Thyme oil was obtained by steam distillation from selected fresh leaves of Thymus zigis L., growing wild in Germany. The EO was provided by HANUS s.r.o. (Slovakia). The p-cymen and thymol contents, which are the most active compounds of thyme oil, were determined as 0.1 and 0.08 mg/kg, respectively. These compounds in the EO were quantified using the high performance liquid chromatography (HPLC) method. Duodenal wall integrity Intestinal integrity was tested by measuring the TEER value. Tissues of duodenal mucosa (0.71 cm2) were incubated at 37°C in chambers with Tyrode’s solution. TEER values were recorded every 3 min over a period of 30 min. The chambers used were constructed similarly to those described by Ussing and Zerhan (1951), with some modifications. They were made from clear acrylic material. The chambers were composed of two symmetrical half-cells each with volume 10.5 ml. A sheet of chicken’s duodenum tissue was mounted between these half-cells. TEER was measured with electrodes made from surgical steel, using a Volt Ohm Meter (MXD-5040RS232 Digital Multimeter with True RMS, METEX Instruments, Korea). Analysis Haemoglobin (Hb) content of blood and total antioxidant status (TAS) were analysed using commercial kits from Randox, UK. To analyse the activities of glutathione peroxidase (GPx, EC 1.11.1.9) in the liver and duodenal mucosa, preweighed pieces of tissue were homogenised in phosphate-buffered saline. Homogenates were centrifuged at 13680 × g at 4°C for 20 min. The enzyme activity in the supernatant as well as the
blood was measured by monitoring oxidation of NADPH + H+ at 340 nm in accordance with Paglia and Valentine (1967), using a commercial kit for the blood (Ransel, Randox, UK). The tissue samples of duodenal mucosa, liver and kidney for malondialdehyde (MDA) measurement were homogenised with de-ionised distilled water and 50 µl of 7.2% butylated hydroxytoluene. The MDA concentrations in tissues were measured using the modified fluorimetric method of Jo and Ahn (1998). Spectrophotometric determination of thioredoxin reductase (TrxR, EC 1.8.1.9) activity was done using the Thioredoxin Reductase Assay Kit (Sigma-Aldrich Inc., St Louis, MO, USA) following the method of Holmgren and Bjornstedt (1995). This is based on the reduction of 5,5′-dithiobis (2-nitrobenzoic) acid (DTNB) with NADPH to 5thio-2-nitrobenzoic acid (TNB), which produces a deep yellow-coloured complex that is measured at 412 nm. The protein concentrations in the examined tissues were measured using the spectrophotometric method published by Bradford (1976). Immunoglobulin A (IgA) in the intestinal mucosa was measured with a Chicken IgA enzyme-linked immunosorbent assay (ELISA Quantitation Set, Bethyl Laboratories, Inc., USA). Intestinal mucosa was prepared using the method described by Nikawa et al. (1999). Phagocytic activity was measured by direct counting procedure using microspheric hydrophilic particles (MSHPs). Ingestion of MSH particles by polymorphonuclear (PMN) cells was determined using a modified test described by Vetvicka et al. (1982). Blood smears were prepared and stained with May-Grünwald and GiemsaRomanowski stains. Phagocytic activity was calculated as the number of white cells containing at least three engulfed particles per 100 white cells (neutrophils) and the index of phagocytic activity was calculated as the number of engulfed particles
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per total number of neutrophils observed. The percentage of phagocytic cells was evaluated using an optical microscope, by counting PMN cells up to 100. The selenium concentrations in diets, blood and tissues were measured using the fluorimetric method of Rodriguez et al. (1994). Dry matter content of diets and tissues was determined by the standard method of drying samples at 105°C.
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Microbiota enumeration Large intestine samples and caecal samples were collected to evaluate selected microbial groups. Samples (1 g) were treated following a standard microbiological method using appropriate dilutions in Ringer solution (1:9, pH 7.0; Oxoid Ltd., Basingstoke, Hampshire, England) in accordance with the International Organization for Standardization (ISO). The appropriate dilutions were plated onto M-Enterococcus agar (Difco Laboratories, Detroit, USA) to identify enterococci, on Mannitol Salt Agar (Difco) for coagulase-negative staphylococci (CoNS), on BairdParker agar supplemented with egg yolk tellurite solution (Becton and Dickinson, Cockeysville, USA) to enumerate coagulase-positive staphylococci (CoPS), including Staphylococcus aureus. The de Mann-Rogosa-Sharpe (MRS) agar was used to detect lactic acid bacteria (LAB). MacConkey agar (Oxoid Ltd., Basingstoke, England) was used to enumerate coliform bacteria, and CLED agar (Biomark) was used to differentiate other Enterobacteriaceae. The plates were incubated at 30°C or 37°C for 24–48 h, depending on the bacterial groups. Bacterial counts were expressed in colony-forming units (log10 CFU) per gram.
per gram of large intestine and caecum content. All microbiological concentrations were subjected to log transformation prior to statistical analysis. Statistical analyses were performed with GraphPadSoftware (USA).
RESULTS The effect of added thyme oil The TEER values were measured in the course of time. They significantly increased over the first 6 min of intestine incubation, when EO was added in the diet (P = 0.0127). After this time, the TEER values were stable up to 30 min. The MDA concentration in duodenal mucosa and kidney significantly decreased in groups of animals with EO in their diets (P = 0.0077 and P = 0.0431, respectively). The IgA concentration in duodenal mucosa as well as phagocytic activity in blood significantly increased in broilers with thyme oil addition in the diets (P = 0.0017 and P < 0.0001, respectively) (Tables 2 and 3). The effect of added selenium Activity of TrxR in the duodenal mucosa, liver and kidney significantly increased in the groups of animals receiving selenium in their diet (P < 0.001, P < 0.0001, P < 0.0001, respectively). Activity of GPx was significantly higher in the blood as well as the liver in both groups with selenium addition (P = 0.0002 and P = 0.0045, respectively). Selenium content significantly increased in blood and duodenal mucosa in both groups of animals with selenium addition in their diet (P < 0.0001 and P < 0.0001, respectively) (Table 2).
Statistical analysis
Bacterial counts
The feeding trial was set up in a randomised block design, with 6 replications of 48 animals (6 cages of 8 broilers each) for each treatment. Statistical analysis of the results used analysis of variance as a 2 × 2 factorial design that represents two main factors: thyme oil (with and without) and selenium (with and without). Three main objectives were examined: the effect of added thyme oil (EO), the effect of added selenium (Se) and the interaction between thyme oil and selenium. Differences between diets with and without additive were analysed by two-way analysis of variance (ANOVA). When interaction between thyme oil × selenium was statistically significant, the simple one-way analysis of variance with post hoc Tukey multiple comparison test was performed. The data presented are the mean values ± SEM. Probability values of less than 0.05 were considered significant. The number of microbial CFUs was expressed as logarithmic (log10) transformation
The bacterial counts in the caecum were slightly influenced in the group with EO and selenium addition by decreases in CoNS (difference 0.74 log cycle), lactic acid bacteria (LAB) (0.30 log cycle), and Enterobacteriaceae (2.15 log cycle). In the large intestine, decrease in Enterobacteriaceae was noted (difference 1.08 log cycle) in the same group (Tables 4 and 5).
DISCUSSION The exact mechanism by which thyme oil influences the various antioxidant parameters is not clear. It is possible that the antioxidant properties of thyme oil are being utilised by the cells, thus sparing the intracellular antioxidant systems (Youdim and Deans, 1999). One of the poultry health-promoting mechanisms of Labiatae plant oils from Origanum
0.33 215.10 0.79 2.59
0.29 265.20 0.71 2.39
0.09 57.00 0.67 2.60
Kidney GPx MDA TrxR Se 0.14 57.51 1.13 2.61
0.46 259.80 1.72 2.63
7.69
0.36 48.32 1.88b 0.75 2.98
2.29 2.34 156.30
Se
0.16 50.19 1.32 2.69
0.42 255.30 1.50 2.72
9.05
0.35 43.27 1.88b 0.98 2.92
2.32 2.11 143.90
EO + Se
0.091 1.925 0.073 0.031
0.018 8.234 0.117 0.063
0.258
0.021 2.271 0.055 0.044 0.055
0.124 0.136 3.702
SEM*
0.4331 0.0431 0.3011 0.1042
1.0000 0.0748 0.5816 0.2306
0.0127
1.0000 0.0077 0.0481 0.0017 0.1200
0.8639 0.8888 0.2713
EO
Se
0.0593 0.6893 < 0.0001 0.8665
0.0045 0.2449 < 0.0001 0.1303
0.5575
0.0557 0.0669 < 0.001 0.1623 < 0.0001
0.0002 0.0638 < 0.0001
a,b Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.001, values are means ± SEM (n = 6), each value represents the mean of 6 cages. *Residual degrees of freedom = 20. BD: basal diet, EO: BD with 0.5 g/kg Thymus vulgaris, Se: BD with 0.4 mg Se/kg DM, EO + Se: BD with 0.5 g/kg Thymus vulgaris and with 0.4 mg Se/kg DM.
0.11 47.55 0.68 2.72
8.72
7.44
0.28 44.64 1.59ab 0.90 2.43
1.46 1.89 119.50
EO
TEER Liver GPx MDA TrxR Se
0.27 60.86 1.34a 0.63 2.62
1.55 1.60 117.30
Blood GPx TAS Se
Duodenal mucosa GPx MDA TrxR IgA Se
BD
Indices
P-value
1.0000 0.7866 0.3508 0.7370
0.3365 0.1321 0.2525 0.6441
0.9313
0.8021 0.1353 0.0481 0.7747 0.4084
0.7320 0.3146 0.1210
interaction
Table 2. Activity of GPx in blood (µkat/g Hb) and tissue (µkat/g protein) and Se concentration in blood (µg/l) and tissue (mg/kg DM), TAS (mmol/l) in plasma, activity of TrxR (µkat/g protein), concentration of MDA(nmol/g protein) in duodenal mucosa, liver and kidney, IgA (mg/g) and TEER (MΩ) in duodenal mucosa of broilers fed on a diet supplemented with 0.5 g/kg thyme essential oil and 0.4 mg Se/kg DM
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DIETARY THYME AND SELENIUM 109
40.50 2.15
PA (%) IPA
43.67 2.35
EO 40.38 2.20
Se 44.00 2.09
EO + Se 0.417 0.039
SEM*
6.54 4.98a 4.47 7.70a 6.52 5.57a
Enterococcus sp. CoNS CoPS LAB Coliforms Enterobacteriaceae
6.45 5.49 4.19 7.63 6.80 4.94
EO 6.37 4.02 3.99 7.48 6.29 3.76
Se 6.23 4.23b 4.26 7.40b 6.42 3.42b
EO + Se
0.067 0.173 0.136 0.083 0.081 0.287
SEM*
0.7491 0.1355 0.9861 0.6618 0.3794 0.2897
EO
0.5885 0.0001 0.4791 0.1978 0.0264 0.0013
Se
P-value
0.8532 0.1561
Se
P-value
0.9445 0.5242 0.3448 0.9767 0.6058 0.7484
Interaction
0.6922 0.0417
Interaction
a,b Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05. Values are means ± SEM (n = 6), each value represents the mean of 6 cages. CoNS: coagulase-negative staphylococci, CoPS: coagulase-positive staphylococci, LAB: lactic acid bacteria; CoNS: a:b (difference 0.74 log cycle), LAB: a:b (difference 0.30 log cycle), Enterobacteriaceae: a:b (difference 2.15 log cycle). *Residual degrees of freedom = 20. BD: basal diet, EO: BD with 0.5 g/kg Thymus vulgaris, Se: BD with 0.4 mg Se/kg DM, EO + Se: BD with 0.5 g/kg Thymus vulgaris and with 0.4 mg Se/kg DM.
BD
Indices (log10 CFU/g)
Table 4. The effect of 0.5 g/kg thyme essential oil and 0.4 mg Se/kg DM on bacterial counts in the caecum of broilers
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Effect of thyme essential oil and selenium on intestine integrity and antioxidant status of broilers a
a
a
a
a
a
I. Placha , J. Takacova , M. Ryzner , K. Cobanova , A. Laukova , V. Strompfova , K. b
Venglovska & S. Faix a
a
Institute of Animal Physiology, SAS, Kosice, Slovak Republic
b
Faculty of Science, Institute of Biology and Ecology, Pavol Jozef Safarik University, Kosice, Slovak Republic Accepted author version posted online: 08 Jan 2014.Published online: 16 Apr 2014.
To cite this article: I. Placha, J. Takacova, M. Ryzner, K. Cobanova, A. Laukova, V. Strompfova, K. Venglovska & S. Faix (2014) Effect of thyme essential oil and selenium on intestine integrity and antioxidant status of broilers, British Poultry Science, 55:1, 105-114, DOI: 10.1080/00071668.2013.873772 To link to this article: http://dx.doi.org/10.1080/00071668.2013.873772
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
British Poultry Science, 2014 Vol. 55, No. 1, 105–114, http://dx.doi.org/10.1080/00071668.2013.873772
Effect of thyme essential oil and selenium on intestine integrity and antioxidant status of broilers I. PLACHA, J. TAKACOVA, M. RYZNER, K. COBANOVA, A. LAUKOVA, V. STROMPFOVA, K. VENGLOVSKA1 AND S. FAIX
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Institute of Animal Physiology, SAS, Kosice, Slovak Republic, and 1Faculty of Science, Institute of Biology and Ecology, Pavol Jozef Safarik University, Kosice, Slovak Republic
Abstract 1. This study evaluated the duodenal wall integrity, antioxidant status as well as some immunological parameters of broiler chickens supplemented with 0.5 g Thymus vulgaris essential oil (EO)/kg diet and 0.4 mg Se/kg DM (dry matter) derived from sodium selenite. 2. A total of 192 one-d-old randomly divided chickens of both sexes (Ross 308 hybrid broilers) were divided into 4 treatment groups of 48 birds each. 3. The first group was fed on a nutritionally balanced basal diet (BD). The other three groups received BD supplemented with 0.5 g/kg thyme oil, or 0.4 mg Se/kg DM, or both feed additives together. 4. The results for the evaluated feed additives were (1) thyme oil – decreased malondialdehyde (MDA) concentration in duodenal mucosa and kidney, increased immunoglobulin A (IgA) concentration in duodenal mucosa, stimulated phagocytic activity in blood, improved intestinal barrier integrity (2) selenium – increased glutathione peroxidase (GPx) activity in blood and liver as well as thioredoxin reductase (TrxR) activity in duodenal mucosa, liver and in the kidney, (3) EO with selenium – increased thioredoxin reductase (TrxR) activity in duodenal mucosa. 5. These results demonstrated that thyme oil alone showed more effective potential to improve intestinal barrier integrity and antioxidant status as well as evoking an immune response in chickens, than if diets were supplemented with both thyme oil and selenium.
INTRODUCTION Since antibiotics, as feed additives, were banned in the European Union in 2006, research into alternatives has gained in importance. Besides probiotic microbiota and synthetic antioxidants, phytogenic substances are most commonly used for the same objective. The market for plant-based performance enhancers has increased since the 1990s. For example, sales of essential oils (EOs) in the EU amounted to 90 t in 1996, while only 10 years later, they reached 600 t (Greathead, 2003). Synthetic antioxidants are currently approved for controlling lipid oxidation in foods, but there is consumer concern over their use (Botterweck et al., 2000). Much attention is now given to the
application of natural antioxidants for stabilising foods against oxidation (Lim et al., 2001). Gut health is a major topic for research, not only in humans but also in animals. Anything that affects the health of the gut will undoubtedly influence the animal as a whole, and consequently alter its nutrient uptake and requirements (Choct, 2009). Hydrogen peroxide is constantly generated within all cell types, including gastric epithelial cells, and has been implicated as a mediator of gastrointestinal injury. Gastric epithelial monolayers, which are exposed to reactive oxygen species (ROS), may cause a significant decrease in trans-epithelial electrical resistance (TEER), which means the transport of ions across the epithelium. When this transport is restricted, the
Correspondence to: I. Placha, Institute of Animal Physiology, Slovak Academy of Sciences, Soltesova 4, 04 01 Kosice, Slovak Republic. E-mail: [email protected] Accepted for publication 18 October 2013.
© 2014 British Poultry Science Ltd
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electrical potential gradient increases across the epithelia. The intestinal barrier is important in the pathogenesis of various diseases. To elucidate passage routes and mechanisms involved in intestinal absorption and permeation, a widely used tool is the Ussing chamber, which was first described in 1951 (Ussing and Zerhan, 1951). EOs are able to scavenge some free radicals, thus playing an important role in prevention of some diseases such as brain dysfunction, cancer, heart disease and immune system decline. Increasing evidence suggests that these diseases may result from cellular damage caused by free radicals. In addition to their ability to scavenge free radicals, there is also evidence that some EOs develop anti-inflammatory activity. For example, chamomile EO has been used for centuries because of its anti-inflammatory properties (Kamatou and Viljoen, 2010). The anti-inflammatory activity of EOs may be attributed not only to their antioxidant activities, but also to their interaction with signalling cascades involving cytokines and regulatory transcription factors, and on the expression of proinflammatory genes (Miguel, 2010). We found in our previous experiment that thyme oil at a concentration of 0.5 g/kg DM (dry matter) can sufficiently control the release of ROS from neutrophils, and by this way, it could simultaneously stimulate neutrophils thus improving phagocytic activity (Placha et al., 2013). Selenium is an essential micro-mineral element with numerous functions such as regulating metabolism, improving immunity, enhancing reproductive performance, preventing cancer and resisting free radicals (Yang et al., 2009). This essential nutrient has a special function in antioxidant control mechanisms as an essential component of the active centre of selenoenzymes (Zuberbuehler et al., 2006). The routine source of Se used to supplement animal feeds is sodium selenite. However, this inorganic source of selenium has been shown to provide relatively low selenium retention (Boldižárová et al., 2005). Even though organic selenium is a superior source because of its active absorption from the intestine in comparison with passive diffusion of sodium selenite from the intestinal tract, inorganic sodium selenite is still used as the principal source of selenium in animal feeds (Mahan, 1995; Schrauzer, 2001; Edens et al., 2002). Brenes and Roura (2010) stated that synergistic effects between EOs and other feed additives could be exploited for maximisation of their antioxidant properties, antibacterial activity and ability to stimulate the immune system. These interactions need to be investigated. The antioxidant activities and role of EOs as well as organic and inorganic forms of selenium in the immune system of chickens are well documented, but their synergism or antagonism has
received little interest to date. Therefore, the aim of our study was to examine whether the administration of Thymus vulgaris EO and selenium alone or together would have a stronger effect on intestinal integrity, parameters of antioxidant status as well as on some immunological parameters in broiler chickens. In addition, their influence on selected microbiota was investigated.
MATERIALS AND METHODS Animal care and use The experiment was carried out in accordance with the established standards for use of animals. The protocol was approved by the Ethical Commission of the Institute of Animal Physiology, Slovak Academy of Sciences in Košice, Slovakia and by Slovak governmental authority (Č.k. RO-820/10-221).
Experimental design and housing A total of 192 Ross 308 hybrid broilers of both sexes was randomly divided at the day of hatching into 4 dietary treatments of 48 birds each. Each dietary treatment consisted of 6 replicates. A replicate was a cage with 8 birds. All cages were placed in the same room, in which temperature was controlled during the experiment. Chickens were reared with a lighting regimen of 23 h of light and 1 h of darkness. The initial room temperature of 32–33°C was reduced weekly by 3°C to a final temperature of 23°C. All birds had free access to water and feed. Animals were fed for 5 weeks on the 4 experimental diets. The first group was given the basal diet (BD, BIOFED – HYD-02, Kolárovo, Slovakia, Table 1); the second was fed on the same BD enriched with 0.5 g/kg of T. vulgaris EO. The third group received BD supplemented with 0.4 mg Se/kg DM derived from sodium selenite (SS), and the 4th was fed on BD enriched with both EO and SS together. The mean analysed values of the Se content in the diets [starter (0–7 d), grower (8–21 d) and finisher (22– 35 d)] for groups 1, 2, 3 and 4 were 0.21 ± 0.01, 0.20 ± 0.03, 0.56 ± 0.05 and 0.60 ± 0.07 mg/kg DM, respectively. At the age of 5 weeks, 24 chickens from each treatment (4 animals/replicate) in the best physical and health condition were sacrificed. Samples of liver, kidney and duodenal mucosa tissues were collected for biochemical analysis and stored at – 70°C until analysis. Their duodenum was separated to measure TEER in vitro. Blood samples for analyses were collected into heparinised tubes.
DIETARY THYME AND SELENIUM
Table 1.
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Ingredients and chemical composition of the basal broiler diet
Ingredients
g/kg
Composition
g/kg
Wheat, ground Maize, ground Soya bean meal, extracted Fish meal Blood meal Limestone Monocalcium phosphate Sodium chloride Premix 1 Sunflower oil
340 400 180 20 10 10 8 3 19 10
Dry matter Crude protein Ash Crude fibre Calcium Phosphorus Lysine Methionine Methionine + cystine Linoleic acid Calculated MEn (N-adjusted metabolisable energy, MJ/kg)
891 190 50 35 7.0 5.0 9.5 4.0 7.5 10.0 12.0
Crude protein, dry matter and selenium are analysed data. The analysed selenium content of the BD was 0.2 mg/kg DM. 1The vitamin/mineral premix provided per kg of complete diet: retinyl acetate: 3.6 mg; cholecalciferol: 0.1 mg; menadione: 4 mg; tocopherol: 100 mg; thiamine: 3 mg; riboflavin: 9 mg; niacin: 60 mg; pantothenic acid: 15 mg; pyridoxine: 6 mg; cyanocobalamin: 0.04 mg; biotin: 0.2 mg; folic acid: 2 mg; lysine: 9.5 g; methionine: 4.0 g; zinc: 100 mg; iodine: 1 mg; cobalamin: 0.4 mg; manganese: 110 mg; copper: 15 mg; iron: 120 mg.
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Thyme oil used in the model experiment Thyme oil was obtained by steam distillation from selected fresh leaves of Thymus zigis L., growing wild in Germany. The EO was provided by HANUS s.r.o. (Slovakia). The p-cymen and thymol contents, which are the most active compounds of thyme oil, were determined as 0.1 and 0.08 mg/kg, respectively. These compounds in the EO were quantified using the high performance liquid chromatography (HPLC) method. Duodenal wall integrity Intestinal integrity was tested by measuring the TEER value. Tissues of duodenal mucosa (0.71 cm2) were incubated at 37°C in chambers with Tyrode’s solution. TEER values were recorded every 3 min over a period of 30 min. The chambers used were constructed similarly to those described by Ussing and Zerhan (1951), with some modifications. They were made from clear acrylic material. The chambers were composed of two symmetrical half-cells each with volume 10.5 ml. A sheet of chicken’s duodenum tissue was mounted between these half-cells. TEER was measured with electrodes made from surgical steel, using a Volt Ohm Meter (MXD-5040RS232 Digital Multimeter with True RMS, METEX Instruments, Korea). Analysis Haemoglobin (Hb) content of blood and total antioxidant status (TAS) were analysed using commercial kits from Randox, UK. To analyse the activities of glutathione peroxidase (GPx, EC 1.11.1.9) in the liver and duodenal mucosa, preweighed pieces of tissue were homogenised in phosphate-buffered saline. Homogenates were centrifuged at 13680 × g at 4°C for 20 min. The enzyme activity in the supernatant as well as the
blood was measured by monitoring oxidation of NADPH + H+ at 340 nm in accordance with Paglia and Valentine (1967), using a commercial kit for the blood (Ransel, Randox, UK). The tissue samples of duodenal mucosa, liver and kidney for malondialdehyde (MDA) measurement were homogenised with de-ionised distilled water and 50 µl of 7.2% butylated hydroxytoluene. The MDA concentrations in tissues were measured using the modified fluorimetric method of Jo and Ahn (1998). Spectrophotometric determination of thioredoxin reductase (TrxR, EC 1.8.1.9) activity was done using the Thioredoxin Reductase Assay Kit (Sigma-Aldrich Inc., St Louis, MO, USA) following the method of Holmgren and Bjornstedt (1995). This is based on the reduction of 5,5′-dithiobis (2-nitrobenzoic) acid (DTNB) with NADPH to 5thio-2-nitrobenzoic acid (TNB), which produces a deep yellow-coloured complex that is measured at 412 nm. The protein concentrations in the examined tissues were measured using the spectrophotometric method published by Bradford (1976). Immunoglobulin A (IgA) in the intestinal mucosa was measured with a Chicken IgA enzyme-linked immunosorbent assay (ELISA Quantitation Set, Bethyl Laboratories, Inc., USA). Intestinal mucosa was prepared using the method described by Nikawa et al. (1999). Phagocytic activity was measured by direct counting procedure using microspheric hydrophilic particles (MSHPs). Ingestion of MSH particles by polymorphonuclear (PMN) cells was determined using a modified test described by Vetvicka et al. (1982). Blood smears were prepared and stained with May-Grünwald and GiemsaRomanowski stains. Phagocytic activity was calculated as the number of white cells containing at least three engulfed particles per 100 white cells (neutrophils) and the index of phagocytic activity was calculated as the number of engulfed particles
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per total number of neutrophils observed. The percentage of phagocytic cells was evaluated using an optical microscope, by counting PMN cells up to 100. The selenium concentrations in diets, blood and tissues were measured using the fluorimetric method of Rodriguez et al. (1994). Dry matter content of diets and tissues was determined by the standard method of drying samples at 105°C.
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Microbiota enumeration Large intestine samples and caecal samples were collected to evaluate selected microbial groups. Samples (1 g) were treated following a standard microbiological method using appropriate dilutions in Ringer solution (1:9, pH 7.0; Oxoid Ltd., Basingstoke, Hampshire, England) in accordance with the International Organization for Standardization (ISO). The appropriate dilutions were plated onto M-Enterococcus agar (Difco Laboratories, Detroit, USA) to identify enterococci, on Mannitol Salt Agar (Difco) for coagulase-negative staphylococci (CoNS), on BairdParker agar supplemented with egg yolk tellurite solution (Becton and Dickinson, Cockeysville, USA) to enumerate coagulase-positive staphylococci (CoPS), including Staphylococcus aureus. The de Mann-Rogosa-Sharpe (MRS) agar was used to detect lactic acid bacteria (LAB). MacConkey agar (Oxoid Ltd., Basingstoke, England) was used to enumerate coliform bacteria, and CLED agar (Biomark) was used to differentiate other Enterobacteriaceae. The plates were incubated at 30°C or 37°C for 24–48 h, depending on the bacterial groups. Bacterial counts were expressed in colony-forming units (log10 CFU) per gram.
per gram of large intestine and caecum content. All microbiological concentrations were subjected to log transformation prior to statistical analysis. Statistical analyses were performed with GraphPadSoftware (USA).
RESULTS The effect of added thyme oil The TEER values were measured in the course of time. They significantly increased over the first 6 min of intestine incubation, when EO was added in the diet (P = 0.0127). After this time, the TEER values were stable up to 30 min. The MDA concentration in duodenal mucosa and kidney significantly decreased in groups of animals with EO in their diets (P = 0.0077 and P = 0.0431, respectively). The IgA concentration in duodenal mucosa as well as phagocytic activity in blood significantly increased in broilers with thyme oil addition in the diets (P = 0.0017 and P < 0.0001, respectively) (Tables 2 and 3). The effect of added selenium Activity of TrxR in the duodenal mucosa, liver and kidney significantly increased in the groups of animals receiving selenium in their diet (P < 0.001, P < 0.0001, P < 0.0001, respectively). Activity of GPx was significantly higher in the blood as well as the liver in both groups with selenium addition (P = 0.0002 and P = 0.0045, respectively). Selenium content significantly increased in blood and duodenal mucosa in both groups of animals with selenium addition in their diet (P < 0.0001 and P < 0.0001, respectively) (Table 2).
Statistical analysis
Bacterial counts
The feeding trial was set up in a randomised block design, with 6 replications of 48 animals (6 cages of 8 broilers each) for each treatment. Statistical analysis of the results used analysis of variance as a 2 × 2 factorial design that represents two main factors: thyme oil (with and without) and selenium (with and without). Three main objectives were examined: the effect of added thyme oil (EO), the effect of added selenium (Se) and the interaction between thyme oil and selenium. Differences between diets with and without additive were analysed by two-way analysis of variance (ANOVA). When interaction between thyme oil × selenium was statistically significant, the simple one-way analysis of variance with post hoc Tukey multiple comparison test was performed. The data presented are the mean values ± SEM. Probability values of less than 0.05 were considered significant. The number of microbial CFUs was expressed as logarithmic (log10) transformation
The bacterial counts in the caecum were slightly influenced in the group with EO and selenium addition by decreases in CoNS (difference 0.74 log cycle), lactic acid bacteria (LAB) (0.30 log cycle), and Enterobacteriaceae (2.15 log cycle). In the large intestine, decrease in Enterobacteriaceae was noted (difference 1.08 log cycle) in the same group (Tables 4 and 5).
DISCUSSION The exact mechanism by which thyme oil influences the various antioxidant parameters is not clear. It is possible that the antioxidant properties of thyme oil are being utilised by the cells, thus sparing the intracellular antioxidant systems (Youdim and Deans, 1999). One of the poultry health-promoting mechanisms of Labiatae plant oils from Origanum
0.33 215.10 0.79 2.59
0.29 265.20 0.71 2.39
0.09 57.00 0.67 2.60
Kidney GPx MDA TrxR Se 0.14 57.51 1.13 2.61
0.46 259.80 1.72 2.63
7.69
0.36 48.32 1.88b 0.75 2.98
2.29 2.34 156.30
Se
0.16 50.19 1.32 2.69
0.42 255.30 1.50 2.72
9.05
0.35 43.27 1.88b 0.98 2.92
2.32 2.11 143.90
EO + Se
0.091 1.925 0.073 0.031
0.018 8.234 0.117 0.063
0.258
0.021 2.271 0.055 0.044 0.055
0.124 0.136 3.702
SEM*
0.4331 0.0431 0.3011 0.1042
1.0000 0.0748 0.5816 0.2306
0.0127
1.0000 0.0077 0.0481 0.0017 0.1200
0.8639 0.8888 0.2713
EO
Se
0.0593 0.6893 < 0.0001 0.8665
0.0045 0.2449 < 0.0001 0.1303
0.5575
0.0557 0.0669 < 0.001 0.1623 < 0.0001
0.0002 0.0638 < 0.0001
a,b Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.001, values are means ± SEM (n = 6), each value represents the mean of 6 cages. *Residual degrees of freedom = 20. BD: basal diet, EO: BD with 0.5 g/kg Thymus vulgaris, Se: BD with 0.4 mg Se/kg DM, EO + Se: BD with 0.5 g/kg Thymus vulgaris and with 0.4 mg Se/kg DM.
0.11 47.55 0.68 2.72
8.72
7.44
0.28 44.64 1.59ab 0.90 2.43
1.46 1.89 119.50
EO
TEER Liver GPx MDA TrxR Se
0.27 60.86 1.34a 0.63 2.62
1.55 1.60 117.30
Blood GPx TAS Se
Duodenal mucosa GPx MDA TrxR IgA Se
BD
Indices
P-value
1.0000 0.7866 0.3508 0.7370
0.3365 0.1321 0.2525 0.6441
0.9313
0.8021 0.1353 0.0481 0.7747 0.4084
0.7320 0.3146 0.1210
interaction
Table 2. Activity of GPx in blood (µkat/g Hb) and tissue (µkat/g protein) and Se concentration in blood (µg/l) and tissue (mg/kg DM), TAS (mmol/l) in plasma, activity of TrxR (µkat/g protein), concentration of MDA(nmol/g protein) in duodenal mucosa, liver and kidney, IgA (mg/g) and TEER (MΩ) in duodenal mucosa of broilers fed on a diet supplemented with 0.5 g/kg thyme essential oil and 0.4 mg Se/kg DM
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DIETARY THYME AND SELENIUM 109
40.50 2.15
PA (%) IPA
43.67 2.35
EO 40.38 2.20
Se 44.00 2.09
EO + Se 0.417 0.039
SEM*
6.54 4.98a 4.47 7.70a 6.52 5.57a
Enterococcus sp. CoNS CoPS LAB Coliforms Enterobacteriaceae
6.45 5.49 4.19 7.63 6.80 4.94
EO 6.37 4.02 3.99 7.48 6.29 3.76
Se 6.23 4.23b 4.26 7.40b 6.42 3.42b
EO + Se
0.067 0.173 0.136 0.083 0.081 0.287
SEM*
0.7491 0.1355 0.9861 0.6618 0.3794 0.2897
EO
0.5885 0.0001 0.4791 0.1978 0.0264 0.0013
Se
P-value
0.8532 0.1561
Se
P-value
0.9445 0.5242 0.3448 0.9767 0.6058 0.7484
Interaction
0.6922 0.0417
Interaction
a,b Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05. Values are means ± SEM (n = 6), each value represents the mean of 6 cages. CoNS: coagulase-negative staphylococci, CoPS: coagulase-positive staphylococci, LAB: lactic acid bacteria; CoNS: a:b (difference 0.74 log cycle), LAB: a:b (difference 0.30 log cycle), Enterobacteriaceae: a:b (difference 2.15 log cycle). *Residual degrees of freedom = 20. BD: basal diet, EO: BD with 0.5 g/kg Thymus vulgaris, Se: BD with 0.4 mg Se/kg DM, EO + Se: BD with 0.5 g/kg Thymus vulgaris and with 0.4 mg Se/kg DM.
BD
Indices (log10 CFU/g)
Table 4. The effect of 0.5 g/kg thyme essential oil and 0.4 mg Se/kg DM on bacterial counts in the caecum of broilers
