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INTRODUCTION The use of antibiotics as growth promoters in broiler
feed has led to concerns about development of antimi-crobial resistance (Castanon, 2007). Since the ban on antibiotic feed additives in the European Union, re-search in alternative substances has gained in impor-tance. In particular for growing broilers, several feed additives have been investigated to increase general health and performance. Besides prebiotics, probiotics, and organic acids, phytogenic substances are also com-monly used for this purpose. As a result, new commer-cial additives derived from plants, including aromatic plant extracts and their purified constituents, have
been examined. Such products have several advantag-es over used commercial antibiotics because they are generally recognized as safe and commonly used items in the food industry (Varel, 2002). These botanicals have received increased attention as possible growth performance enhancers for animals in the last decade via their beneficial influence on lipid metabolism, and antimicrobial and antioxidant properties (Botsoglou et al., 2002), ability to stimulate digestion (Hernandez et al., 2004), immune enhancing activity, and antiinflam-matory potential (Acamovic and Brooker, 2005). Many studies have been reported on the supplementation of poultry diets with some essential oils that enhanced weight gain, improved carcass quality, and reduced mortality rates (Williams and Losa, 2001). These char-acteristics are possibly related to the function of their compounds. In general, thymol [5-methyl-2-(1-methyle-thyl) phenol], a main component of thyme essential oil, and its isomer, carvacrol [2-methyl-5-(1-methylethyl) phenol], a main component of oregano essential oil, are
Effect of thymol and carvacrol feed supplementation on performance, antioxidant enzyme activities, fatty acid composition, digestive enzyme
activities, and immune response in broiler chickens
H. Hashemipour ,*1 H. Kermanshahi ,* A. Golian ,* and T. Veldkamp †
* Excellence Centre for Animal Science and Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, PO Box 91775-1163, Mashhad, Iran; and † Wageningen UR Livestock Research,
PO Box 65, NL-8200 AB Lelystad, the Netherlands
ABSTRACT This trial was conducted to evaluate the effects of dietary supplementation of phytogenic prod-uct containing an equal mixture of thymol and carva-crol at 4 levels (0, 60, 100, and 200 mg/kg of diet) on performance, antioxidant enzyme activities, fatty acid composition, digestive enzyme activities, and immune response in broiler chickens. Each of the 4 diets was fed to 5 replicates of 12 chicks each from d 0 to 42. The inclusion of thymol + carvacrol linearly decreased (P< 0.05) feed intake, but the highest (P < 0.05) BW gain (ADG) and feed efficiency was observed in broilers offered 200 mg/kg of phytogenic product. The phyto-genic product linearly increased (P < 0.05) superoxide dismutase and glutathione peroxidase activities and decreased (P < 0.05) malondialdehyde level in thigh muscle at d 42 and serum and liver at d 24 and 42. To-tal saturated fatty acids were depressed (P < 0.05) and
total polyunsaturated fatty acid and n-6 were linearly increased (P < 0.05) in serum and thigh by the inclu-sion of phytogenic product compared with the control diet. Supplementation with thymol + carvacrol also in-creased intestinal and pancreatic trypsin, lipase, and protease activities in 24-d-old (linear, P < 0.05) but not in 42-d-old birds. Thymol + carvacrol modified (linear, P < 0.05) immune response by increasing hypersensi-tivity response, total and IgG anti-sheep red blood cell titers, and decreasing heterophil to lymphocyte ratio compared with the control group. However, hemato-logical parameters and lymphoid organ weight were not affected by thymol + carvacrol. Thus, feed supplemen-tation with thymol + carvacrol enhanced performance, increased antioxidant enzyme activities, retarded lipid oxidation, enhanced digestive enzyme activities, and improved immune response of broilers.
Key words: phytogenic product , antioxidant enzyme , digestive enzyme , immunity , broiler
2013 Poultry Science 92 :2059–2069http://dx.doi.org/ 10.3382/ps.2012-02685
METABOLISM AND NUTRITION
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Received August 15, 2012. Accepted January 14, 2013. 1 Corresponding author: [email protected] or
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principally responsible for these activities (Yanishlieva et al., 1999).
Thymol and carvacrol are reported to inhibit lipid peroxidation. Lipid peroxidation is an autocatalytic mechanism leading to oxidative destruction of cellu-lar membranes (Rhee et al., 1996). The destruction can lead to cell death and also to the production of toxic and reactive aldehyde metabolites, known as free radicals. Among these free radicals, malondialdehyde (MDA) is the most important and main final product of lipid peroxidation that has often been used for de-termining oxidative damage (Jensen et al., 1997). Thy-mol and carvacrol both have strong antioxidant activity (Yanishlieva et al., 1999). Oregano added in doses of 50 to 100 mg/kg to the diet of chickens exerted an an-tioxidant effect in the broiler tissues (Botsoglou et al., 2002). A dietary supply of thyme oil or thymol to ag-ing rats showed a beneficial effect on the antioxidative enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), as well as on polyunsaturated fatty acid (PUFA) composition in various tissues. Rats fed these supplements had greater levels of SOD and GSH-PX and more concentration of PUFA in phospho-lipids of the brain than the untreated controls (Youdim and Deans, 2000).
Thymol and carvacrol are also believed to exhibit a range of beneficial physiological effects. Thymol has been reported to stimulate digestive secretions such as salivary amylase in humans and bile acids, gastric, pan-creatic enzymes (i.e. lipase, amylase, and proteases), and intestinal mucosa in rats (Platel and Srinivasan, 2004). A significant increase in pancreatic trypsin, am-ylase, and maltase activities in broilers fed different blends of commercial essential oils has been reported as well (Jang et al., 2007). However, Lee et al. (2003) showed no clear effects on enzyme activities in chickens fed dietary thymol and carvacrol after 21 or 40 d of age.
The immune status of the host is known to play an important role in resistance to various infections and essential oils, and extracts may enhance cellular and humoral responses of broiler chickens. Therefore, they could play important roles in strengthening the defense system of birds against invasion by infectious organ-isms (Acamovic and Brooker, 2005). However, the im-mune mechanisms affected by the essential oils have not been thoroughly investigated in chickens.
Therefore, this study was conducted to determine the effects of dietary supplementation of phytogenic prod-uct containing an equal mixture of thymol and car-vacrol on performance, antioxidant enzyme activities, fatty acid composition, digestive enzyme activities, and immune response in broilers.
MATERIALS AND METHODS
Birds, Diets, and ManagementThis study was carried out using a total of 240 Ross-
308 male broiler chicks, following the protocols of Ani-
mal Care Committee of the Ferdowsi University of Mashhad, Iran. One-day-old chicks were obtained from a local hatchery and divided into 20 groups of 12 birds each. There were 4 treatments including 0, 60, 100, and 200 mg/kg of Next Enhance 150 (1:1 thymol:carvacrol; Novus International Inc., St. Louis, MO). According to the manufacturer, Next Enhance 150 contains 50% ac-tive components, including thymol and carvacrol.
Each diet was randomly fed to 5 groups of chicks. The feeding regimen consisted of a starter (1 to 10 d), grower (11 to 24 d), and finisher diet (25 to 42 d). The basal diet was fed as mash and prepared with the same batch of ingredients for starter, grower, and fin-isher periods and was formulated to meet the nutrient requirements according to Ross-308 rearing guidelines (Aviagen, 2007). All birds had free access to feed and water. The ingredients and chemical composition of the basal diets are shown in Table 1. Next Enhance 150 was added to 100 g of wheat bran and then was blended with premixes. Finally, the premixes were mixed with the basal diet. Feed was prepared weekly and stored in airtight containers. Temperature was initially set at 32°C on d 1 and decreased linearly by 0.5°C per day to a temperature of 21°C. During the study, the birds re-ceived a lighting regimen of 24L:0D from 1 to d 7, and afterward 23L:1D until d 42.
Essential Oil AnalysisTo extract the active components from the feed and
Next Enhance 150, 4 g of grinded feed samples were weighed into a centrifuge tube. The samples were mixed with 2.5 mL of distilled water and 1 mL of ethanol and allowed to stand for 15 min. Then, 12 mL of diethyl ether was added; the samples were shaken for 16 h and centrifuged at 15,000 × g for 5 min at 4°C. The cali-bration samples were prepared from control feed and supplemented with standard solutions of carvacrol and thymol at 5 different concentrations (5, 10, 20, 40, and 100 mg/L in ethanol) or unsupplemented ethanol as a blank.
To analyze the extracts, 1 mL of each supernatant was injected into the gas chromatograph with flame ionization detector. Gas chromatographic analyses were performed using a GC PU 4500 system (Shimadzu Corp., Kyoto, Japan) equipped with a flame ioniza-tion detector and E30 (30 m × 0.32 mm ID, 5% phe-nyl methyl silicone; phase thickness 0.5 mm) capillary column. The column temperature ranged from 80 to 202°C increments of 8°C per minute. Helium was used as the carrier gas at a flow rate of 1.5 mL/min. Sample injection was carried out in splitless mode at 200°C with splitless time of 1 min with a sample injection vol-ume of 0.5 μL. Temperature of the detector was 202°C. Oven temperature was maintained initially at 80°C for 2 min, then raised at a rate of 8°C/min to 125°C and maintained for 10 min, then raised at a rate of 25°C/min to 200°C and maintained for 10 min. The 5 con-centration linear calibration curves were calculated us-
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ing the internal standards. Using the peak heights, the concentrations (mg/kg) of the analysts in the samples were calculated from the calibration curves.
SamplingThe experimental period lasted 42 d. On d 10, 24,
and 42, birds were weighed by pen, and feed consump-tion was recorded. Feed efficiency and ADG were cal-culated for each phase. One bird per pen was selected at random and humanely killed by cervical dislocation for organ sampling (liver, pancreas, spleen, bursa, and thymus) on d 24 and 42. The left breast and left leg of each bird were removed, vacuum packed, and stored at −20°C until antioxidant enzyme analysis.
The pancreas was harvested from each bird within 5 min after death, placed in aluminum foil, snap frozen in liquid nitrogen, and stored at −80°C. Prior to the en-zyme assays, frozen pancreas tissues were homogenized in 0.05 mM Tris buffer (pH 7.4) with 0.05 mM CaCl2 using a tissue homogenizer. The homogenates were cen-trifuged twice (2,420 × g for 5 min, then 16,000 × g for 15 min; both at 4°C). After the second centrifugation, the supernatant was divided into aliquots and stored at −80°C.
Intestinal digesta were immediately removed by gen-tle finger stripping of the intestinal segments. The di-gesta samples were diluted 10× based on the sample weight, with ice-cold PBS (pH 7.0), homogenized for 60 s, and sonicated for 1 min with 3 cycles at 30-s inter-
vals. The samples were then centrifuged at 18,000 × g for 20 min at 4°C. The supernatants were divided into small portions and stored at −80°C for enzyme assays.
Assay of Antioxidant Indices in Serum and Muscle
For biochemical assays, liver and muscle tissues were homogenized in ice-cold isotonic physiological saline to form homogenates at the concentration of 0.1 g/mL. The samples were centrifuged and the already prepared supernatants and sera were subjected to the measurement of SOD and GSH-Px activities and MDA levels by spectrophotometric methods using a spectro-photometer (Leng Guang SFZ1606017568, Shanghai, China). Activity of SOD was measured by the xan-thine oxidase method, which monitors the inhibition of reduction of nitro blue tetrazolium by the sample (Winterbourn et al., 1975). Activity of GSH-Px was de-tected with 5,5′-dithiobis-p-nitrobenzoic acid, and the change of absorbance at 412 nm was monitored using a spectrophotometer (Hafeman et al., 1974). The MDA level was analyzed with 2-TBA, monitoring the change of absorbance at 532 nm with the spectrophotometer (Jensen et al., 1997).
Lipid ExtractionExtraction of lipids from tissue specimens was con-
ducted with the method of Hara and Radin (1978).
Table 1. Chemical composition of basal diets
ItemStarter
(1 to 10 d)Grower
(11 to 24 d)Finisher
(25 to 42 d)
Ingredient (%) Corn 53.20 55.88 57.25 Soybean meal, 44% protein 38.41 34.90 33.31 Wheat bran 2.02 2.02 2.02 Vegetable oil 2.08 3.60 4.10 Limestone 1.30 1.10 1.04 Dicalcium phosphate 1.65 1.40 1.31 Salt 0.42 0.42 0.40 dl-Met 0.15 0.10 0.07 HCl-Lys 0.21 0.08 0.00 Thr 0.06 0.00 0.00 Vitamin premix1 0.25 0.25 0.25 Mineral premix2 0.25 0.25 0.25Calculated composition (%, unless otherwise noted) ME (kcal/kg) 2,850 2,970 3,020 CP 22.1 20.7 19.8 Ca 1.00 0.85 0.80 Available P 0.47 0.42 0.39 Sodium 0.18 0.18 0.17 Lys 1.35 1.17 1.03 Met 0.48 0.42 0.39 Met + Cys 1.01 0.90 0.81 Thr 0.89 0.78 0.70
1Vitamin premix provided the following per kilogram of diet: vitamin A (trans-retinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 3,500 IU; vitamin E (dl-α-tocopheryl acetate), 60 mg; vitamin K (menadione), 3 mg; thiamine, 3 mg; riboflavin, 6 mg; pyridoxine, 5 mg; vitamin B12 (cyanocobalamin), 0.01 mg; niacin, 45 mg; pantothenic acid (d-calcium pantothenate), 11 mg; folic acid, 1 mg; biotin, 0.15 mg; choline chloride, 500 mg; ethoxyquin (antioxidant), 150 mg.
2Mineral premix provided the following per kilogram of diet: Fe, 60 mg; Mn, 100 mg; Zn, 60 mg; Cu, 10 mg; I, 1 mg; Co, 0.2 mg; Se, 0.15 mg.
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Briefly, a 1-g tissue specimen was homogenized in 3:2 (vol/vol) 10-mL hexane-isopropanol mixture for 30 s. Tissue homogenate was centrifuged in 2,260 × g for 10 min at room temperature; supernatant was taken and used for analysis.
Preparation of Fatty Acid Methyl Esters
For preparation of methyl esters, lipid extract in the hexane/isopropanol phase was taken into 30-mL ex-perimental tubes. Five milliliters of 2% methanolic sul-phuric acid was added, and the mixture was vortexed. This mixture was left to methylate at 50°C incubation for 15 h. Then it was cooled at room temperature, and then 5 mL of 5% sodium chloride was added and mixed. The produced fatty acid methyl esters were extracted with 5 mL of hexane. Then the hexane phase was taken using a pipette and treated with 5 mL of 2% KHCO3. Solvent of methyl ester-containing mixture was evapo-rated at 45°C with nitrogen flow and solved with 1 mL of hexane. Then they were taken to closed 2-mL autosampler vials and analyzed by gas chromatography (Christie, 1992).
Determination of Digestive Enzyme Activities
Amylase activity was determined using the method of Somogyi (1960). One unit of amylase activity was defined as the amount of amylase to release 1 mg of glucose in 30 min at 38°C per mg of intestinal digesta protein or pancreas.
Lipase activity was assayed using the method de-scribed by Tietz and Fiereck (1966). Lipase activity unit was equal to the volume (in mL) of 0.05 M NaOH required to neutralize the fatty acid liberated during a 6-h incubation with 3 mL of lipase substrate at 38°C per mg of intestinal digesta protein or pancreas.
Protease activity was analyzed using the method of Lynn and Clevette-Radford (1984). The protease activ-ity unit was defined as milligrams of azocasein degrad-ed during 2 h of incubation at 38°C per mg of intestinal digesta protein or pancreas. Trypsin activity was then measured using benzoyl dl-arginine p-nitroanilide as a substrate according to procedures described by Lainé et al. (1993). One unit of enzyme activity was defined as the trypsin hydrolysis of 1 μmol of substrate in 1 min per mg of intestinal digesta protein or pancreas after activation with 0.1 U/mL of enterokinase.
The intestinal digesta protein concentrations were determined by the method of Lowry et al. (1951). Bo-vine serum albumin was used as a standard.
Immunological Measurements
Cellular immunity was assessed by a cutaneous baso-phil hypersensitivity test in vivo by using phytohemag-glutinin-P. At d 10 (according to Corrier and DeLoach,
1990), the toe web between the third and fourth digits of the right foot was measured in millimeters with a constant-tension micrometer. Immediately after mea-surement, 100 μg of phytohemagglutinin (suspended in 0.10 mL of sterile saline) was injected into the toe web. The toe web swelling was measured 24 and 48 h after injection. The response was determined by subtracting the skin thickness of the first measurement from the skin thickness of the second measurement (Corrier and DeLoach, 1990). At d 28, 2 birds per pen were random-ly selected for a primary antibody response to sheep red blood cell (SRBC). A 1.0-mL suspension (7% vol/vol) of SRBC was injected intraperitoneally. The SRBC was used as an antigen to quantify the antibody response. Blood samples were collected at 7 and 14 d after injec-tion. The serum from each sample was collected, heat inactivated at 56°C for 30 min, and then analyzed for total and IgG (mercaptoethanol-resistant) anti-SRBC antibodies as described by Cheema et al. (2003).
At 40 d of age, 2 birds per replicate were selected and their blood samples were collected using heparin-containing syringes to avoid blood clot formation for hematological analysis. Blood smears were prepared on slides and painted by Giemsa method. One hundred leukocytes per sample were counted by heterophil to lymphocyte separation under an optical microscope, and then heterophil to lymphocyte ratio (H/L) was measured. The white blood cell and red blood cell counts were determined by an improved Neubauer he-mocytometer method (Jain, 1986). The hematocrit and hemoglobin values were measured by microhematocrit and colorimetric cyanomethemoglobin methods, re-spectively (Baker and Silverton, 1985).
Immune organ weights were obtained from 2 birds per pen. Birds were weighed and killed. The bursa, spleen, and thymus from the left side of the neck were dissected and weighed immediately. Organ weights were expressed as a percentage of BW.
Statistical AnalysisThe experiment was carried out as a completely ran-
domized design with 4 treatments. Data were analyzed using PROC GLM of SAS (SAS Institute Inc., 2001). Orthogonal polynomial contrasts were used to test the linear and quadratic effects of the increasing levels of supplementation of carvacrol + thymol.
RESULTS AND DISCUSSION
Chemical Composition of Plant Extracts and Diets
The components of Next Enhance 150 were carvacrol (54.13%) and thymol (45.87%). Carvacrol and thymol contents of the experimental diets are shown in Table 2. The terpene concentration in the diet and commer-cial product was determined because terpenes or other compounds were responsible for the observed effects.
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Growth Performance
The effects of dietary thymol + carvacrol supplement on growth performance traits of broilers at different phases are shown in Table 3. At 10 d of age, inclusion of thymol + carvacrol linearly increased (P < 0.01) ADG and G:F compared with birds fed the control diet and did not affect ADFI. From 11 to 24 d of age, thy-mol + carvacrol had significant effects on ADG, ADFI, and G:F; thymol + carvacrol linearly increased ADG and lowered ADFI, but the highest feed efficiency was observed in broilers offered 200 mg/kg of thymol + carvacrol (6.4% more than the control; P < 0.01). From 25 to 42 d of age, thymol + carvacrol supplementa-tion significantly linearly increased ADG (P < 0.01) and G:F (P < 0.01) and linearly decreased ADFI com-pared with the nonsupplemented group. At 42 d of age, thymol + carvacrol linearly increased ADG and G:F with the highest ADG and G:F being obtained with 200 mg/kg of that; however, it decreased (linear, P < 0.01) ADFI compared with the control group. These results were similar to finding of Cross et al. (2007) who supplemented thyme essential oil at a level of 1,000 mg/kg and found an improved BW gain, although FI decreased by almost 10%. Jamroz and Kamel (2002) observed improvements of 8.1% in daily gain and 7.7%
in feed conversion ratios in 17-d-old poults fed a diet supplemented with a plant extract containing carvacrol at 300 mg/kg. Various dietary herbs, plant extracts, and especially essential oils have been studied for their antimicrobial and growth promoter abilities (Cross et al., 2007). The thymol and carvacrol effects on perfor-mance could relate to increased efficiency of feed uti-lization (Lee et al., 2003). Hernandez et al. (2004) ob-served that the effect of different additives containing thymol and carvacrol, pepper essential oils (200 mg/kg), or sage and rosemary extracts (5,000 mg/kg) on digestibility improved the broiler performance. Addi-tionally, the evident antibacterial activity (Botsoglou et al., 2002), the improvement in digestibility (Hernandez et al., 2004) and in feed utilization (Lee et al., 2003), and the digestive and pancreatic enzymes stimulation (Lee et al., 2003) in response to thyme essential oil in-gestion might increase animal performance.
The results obtained from many studies, in which the effects of thymol and carvacrol were investigated on growth performance in poultry, were not consistent. In contrast with this study, dietary supplementation of oregano essential oil to broilers (Botsoglou et al., 2002) at 50 and 100, 150, 300, and 1,000 mg/kg had no beneficial effect on growth performance. Cross et al. (2003) indicated that the inclusion of thyme oil had no
Table 2. Calculated and analyzed carvacrol and thymol contents of the experimental diets (mg/kg)
Experimental diet1
Calculated Analyzed
Carvacrol Thymol Carvacrol Thymol
Control — — — —NE60 32.48 27.52 31.33 26.04NE100 54.13 45.87 51 40.5NE200 108.24 91.74 104.4 87.9
1Control, contained no Next Enhance 150 (Novus International Inc., St. Louis, MO); NE60, 60 mg/kg of Next Enhance 150; NE100, 100 mg/kg of Next Enhance 150; NE200, 200 mg/kg of Next Enhance 150.
Table 3. Effects of dietary thymol + carvacrol supplement on growth performance traits of broilers at different phases
Performance Control
Thymol + carvacrol (mg/kg)
SEM
Probability
Treatment
Dose response1
60 100 200 Lin. Quad.
0 to 10 d ADG (g) 24.7 26.1 26.2 27.2 0.297 0.007 0.009 NS2
ADFI (g) 29.4 29.0 28.6 28.4 0.452 NS NS NS G:F (g/kg) 840 900 917 958 17.55 0.004 0.024 NS11 to 24 d ADG (g) 62.2 63.9 64.3 64.5 0.374 0.003 0.002 0.015 ADFI (g) 94.5 92.8 93.0 92.2 0.348 0.004 0.019 NS G:F (g/kg) 658 689 692 700 3.864 0.001 0.001 0.00425 to 42 d ADG (g) 98.1 100 100 101 0.392 0.007 0.007 NS ADFI (g) 194 192 191 190 0.411 0.004 0.003 NS G:F (g/kg) 507 522 525 533 2.775 0.002 0.002 NS0 to 42 d ADG (g) 68.6 70.4 70.6 71.4 0.168 0.001 0.001 0.002 ADFI (g) 121 120 120 119 0.285 0.003 0.003 NS G:F (g/kg) 565 587 590 601 2.194 0.001 0.001 0.003
1Linear and quadratic effects.2NS, P > 0.05.
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effect on BW gain of broilers. Lee et al. (2003) pointed out that 200 mg/kg of thymol in diet did not affect the BW gain, feed intake, and FCR of female broilers. The reason for the lack of effects of thymol, carvacrol, or both on performance may be related to the composition of the basal diet and environmental conditions (Lee et al., 2003).
Antioxidant Enzyme ActivitiesThe effects of dietary thymol + carvacrol supplement
on SOD and GSH-Px activities and MDA concentration in muscles of broilers at 42 d of age are shown in Table 4. In contrast with the control group, supplementation of diets with thymol + carvacrol linearly elevated (P < 0.01) SOD and GSH-PX activities and depressed (lin-ear, P < 0.05) MDA in thigh muscle. However, the phytogenic product did not affect SOD and GSH-PX activities and MDA level in breast muscle compared with those of the control group. In the present study, thigh muscle exhibited more SOD and GSH-Px activi-ties and greater MDA level than breast muscle. Simi-larly, thigh muscle of chickens fed oregano oil seemed to be more susceptible to oxidation compared with breast muscle (Botsoglou et al., 2002). Susceptibility of meat to lipid oxidation depends on the animal species, mus-cle type, and anatomical location (Rhee et al., 1996). In line with the present study, Renerre et al. (1999) described greater antioxidant enzyme activities in oxi-dative muscles (leg) than in glycolytic muscles (breast) in turkeys. This could be considered as a protective system preventing or delaying the onset of oxidative stress in susceptible muscles. Moreover, according to Fasseas et al. (2007), the greater GSH-Px activity ob-served in legs might be due to a greater content of total and soluble selenium and in particular PUFA found in oxidative muscles. Selenium is a component of enzyme GSH-Px which prevents free radical formation that is very harmful to cells by way of disrupting cell integ-rity (Kanacki et al., 2008). Considering that MDA is produced as a result of PUFA oxidation, differences in the effect of the same antioxidant compounds on
the muscle type could be explained by greater absolute content of PUFA in thigh compared with breast tissue (Jensen et al., 1997).
Antioxidant enzymes including GSH-Px and SOD are synthesized and regulated endogenously. The SOD plays an important role in protecting cells from dam-age caused by reactive oxygen species, but this process requires dietary supply of the appropriate nutrients (Yesilbag et al., 2011). For example, oregano essential oil added in doses of 50 to 100 mg/kg to the diet of chickens exerted an antioxidant effect in the animal tis-sues (Botsoglou et al., 2002). Such antioxidant effects would be expected to improve the health of poultry. From these results, it can be stated that supplementa-tion with the natural antioxidants thymol, carvacrol, or both could be applied in the future to improve the nutritional quality of chicken meat.
The effects of dietary thymol + carvacrol supplement on SOD and GSH-Px activities and MDA concentra-tion in serum and liver of broilers at 24 and 42 d of age are shown in Table 5. In general, compared with the control group, chickens supplemented with thymol + carvacrol showed linear increased (P < 0.05) SOD and GSH-Px activities in serum at 24 d of age, and also MDA level in serum decreased with the inclusion of thymol + carvacrol at 24 d of age. The inclusion of thy-mol + carvacrol linearly enhanced (P < 0.01) SOD and GSH-PX and reduced (P < 0.01) MDA in serum at 42 d of age compared with the control diet. At d 24, diet treated with thymol + carvacrol increased SOD (linear, P < 0.01) and GSH-PX (quadratic, P < 0.01) activities in liver. The MDA level in liver was linearly reduced (P < 0.01) by the inclusion of thymol + carvacrol at d 24. Inclusion of phytogenic product elevated SOD linear and GSH-PX quadratic and reduced (linear, P < 0.01) MDA level at d 42. The serum and liver SOD and GSH-PX activity results obtained in the present study imply that the active substances of the phytogenic product may improve the antioxidative status of broilers due to the antioxidant property of thymol and carvacrol by elevating the activity of antioxidant enzymes. Yan-ishlieva et al. (1999) discussed the relationship between
Table 4. Effects of dietary thymol + carvacrol supplement on superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) concentration in muscles of broilers at 42 d of age
Oxidative status Control
Thymol + carvacrol (mg/kg)
SEM
Probability
Treatment
Dose response1
60 100 200 Lin. Quad.
Thigh muscle SOD (U/mg of protein) 138 141 150 154 0.792 0.001 0.004 NS2
GSH-PX (U/mg of protein) 1.82 2.09 2.16 2.20 0.073 0.080 0.006 0.046 MDA (nmol/mg of protein) 4.28 3.54 3.51 2.71 0.166 0.001 0.027 NSBreast muscle SOD (U/mg of protein) 134 134 136 135 0.514 NS NS NS GSH-PX (U/mg of protein) 1.40 1.39 1.40 1.42 0.013 NS NS NS MDA (nmol/mg of protein) 1.29 1.29 1.29 1.28 0.050 NS NS NS
1Linear and quadratic effects.2NS, P > 0.05.
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the antioxidant property and the chemical composition of essential oils. It was suggested that the high anti-oxidant activity of thymol is due to the presence of phenolic OH groups that act as hydrogen donors to the proxy radicals produced during the first step in lipid oxidation, thus retarding the hydroxyl peroxide forma-tion. Lin et al. (2003) reported that the intake of herbs in chickens results in an increase in serum antioxidant enzyme activities and a decrease in MDA level.
Based on these findings, we state that thymol and carvacrol might play an important role as an exogenous antioxidant and could also be applicable as a protec-tive agent against tissue damage. Thymol and carvacrol seem to have similar effectiveness, according to the def-inition of Yanishlieva et al. (1999), that the possibility of blocking the radical chain process by interaction with peroxide radicals is similar in both compounds. How-ever, they probably differ in the mechanism of action on broiler meat deterioration because their molecular asymmetries differ. Yanishlieva et al. (1999) also pro-posed that during the oxidation of lipids at high ambi-ent temperatures, thymol is a more effective and more active antioxidant than carvacrol, and both compounds differ in the mechanism of their inhibiting action, which depends on the character of the lipid medium. These authors also suggested that thymol is a better antioxi-dant than carvacrol because thymol has greater steric hindrance of the phenolic group than carvacrol.
Fatty Acid Compositions
The effects of dietary thymol + carvacrol on fatty acid composition of serum, thigh, and breast muscles in broilers at 42 d of age are given in Table 6. Addition
of thymol + carvacrol to the diets modified the fatty acid composition of serum and thigh muscle by reduc-ing (linear, P < 0.01) total saturated fatty acid (SFA) and increasing (linear, P < 0.01) total PUFA and n-6 in serum and thigh and increasing (linear, P < 0.01) total monounsaturated fatty acids in the thigh. The n-3 in serum and thigh was not affected by the inclusion and dose of the feed additive. Fatty acid composition in breast was not influenced by any levels of this feed additive. The PUFA are the most sensitive fractions to oxidation processes and lipid oxidation in meat and are one of the reasons for quality degradation during storage. Enhancement of unsaturated fatty acids in thigh lipids would result from diminution of fatty acid oxidation in thigh. This antioxidant activity of thymol and carvacrol was supported in the present study; the PUFA concentration in serum and thigh meat was sig-nificantly greater than those in control birds. It was thought that the antioxidant activity of thymol and carvacrol blocked lipid peroxidation of thigh lipids, especially PUFA. For this reason, PUFA in the thigh and serum in birds fed diets supplemented with thymol + carvacrol increased linearly compared with control birds.
Animals receiving thymol had greater antioxidant en-zyme activities and greater concentration of PUFA in phospholipids of the brain than the untreated control (Youdim and Deans, 2000). Ciftci et al. (2010) sug-gested that cinnamon oil may increase PUFA ratio and decrease SFA content in serum and meat lipids because of the hypolipidemic and antioxidant properties of cin-namon oil in diets. Ertas et al. (2005) reported that the supplementation of coriander sativum modified carcass lipid composition of quails by lowering SFA proportions and enhancing PUFA (particularly n-3) contents.
Table 5. Effects of dietary thymol + carvacrol supplement on superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) concentration in serum and liver of broilers at 24 and 42 d of age
Oxidative status Control
Thymol + carvacrol (mg/kg)
SEM
Probability
Treatment
Dose response1
60 100 200 Lin. Quad.
Serum 24 d SOD (U/mL) 143 144 162 163 1.965 0.001 0.017 NS2
GSH-PX (U/mL) 169 175 180 181 2.433 0.016 0.026 NS MDA (nmol/mL) 6.56 6.47 5.59 5.48 0.190 0.001 NS NS 42 d SOD (U/mL) 144 157 162 168 3.233 0.010 0.004 NS GSH-PX (U/mL) 170 181 184 188 2.339 0.021 0.002 0.042 MDA (nmol/mL) 7.61 6.81 6.50 6.28 0.197 0.011 0.003 0.042Liver 24 d SOD (U/mL) 294 299 309 316 1.940 0.001 0.008 NS GSH-PX (U/mL) 2.22 2.56 2.48 2.49 0.038 0.001 0.002 0.001 MDA (nmol/mL) 4.93 4.13 3.62 3.04 0.150 0.001 0.002 NS 42 d SOD (U/mL) 288 311 316 317 1.143 0.001 0.001 0.001 GSH-PX (U/mL) 3.14 3.66 3.63 3.69 0.028 0.001 0.001 0.001 MDA (nmol/mL) 5.15 4.68 4.42 4.22 0.153 0.034 0.013 NS
1Linear and quadratic effects.2NS, P > 0.05.
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Digestive Enzyme Activities
The effects of dietary thymol + carvacrol on intesti-nal and pancreatic digestive enzyme activities in broil-ers (U/mg of digesta protein) at 24 and 42 d of age are shown in Table 7. Birds fed diets supplemented with the phytogenic product produced greater (linear, P < 0.05) activities of intestinal trypsin, lipase, and prote-ase compared with those in control birds at 24 d of age. Compared with the control diets, treatments did not have a significant effect on digestive enzyme activities at 42 d of age.
For pancreatic measurement, phytogenic product lin-early increased activities of pancreatic trypsin, lipase, and protease compared with that of control group at 24 d of age. The pancreatic digestive enzyme activities were not influenced in birds fed thymol + carvacrol supplemented diet at d 42. From the present results, it may be postulated that the supplementation of phyto-genic product would trigger the secretion of digestive enzymes under certain circumstances (e.g., age of birds, dose of phytogenic, bird species, type and quality of basal diet, bird health, and environmental and man-agement conditions), which could enhance digestion of nutrients in the intestine.
In agreement with the present study, commercial CRINA containing 29% of active components, includ-ing thymol, significantly increased trypsin activity at d 21 but not at d 40 in broilers (Lee et al., 2003). In another study (Jang et al., 2007) in which thymol was used, pancreatic total and activities of trypsin and to-tal lipase activity were significantly greater in 50 mg of thymol than those in the control diet. It has been re-ported that feeding essential oil, extracted from herbs,
improved the secretion of pancreatic digestive enzymes in broiler chickens (Jang et al., 2007). A study with broilers demonstrated that a blend of commercial essen-tial oil components stimulated activities and secretion of digestive enzymes including protease and amylase compared with a control group (Williams and Losa, 2001). A mixture of carvacrol, cinnamaldehyde, and capsaicin used as feed additive for broilers is shown to enhance activities of pancreatic trypsin and α-amylase in tissue, as well as in the jejunal chyme content (Jang et al., 2007).
Immune ResponseThe effects of dietary thymol + carvacrol on hy-
persensitivity and antibody production in broilers are shown in Table 8. Continuous application of thymol + carvacrol has the potential to increase the cellular and humoral immune responses. Phytohaemagglutinin-P in-jection linearly increased (P < 0.01) toe web thickness within 24 and 48 h after injection in all experimental birds compared with the control group. The phytogenic product linearly increased (P < 0.01) the primary re-sponse against SRBC antigen and IgG; also, secondary response and IgG were enhanced (P < 0.05) in birds fed diets containing thymol + carvacrol.
In poultry production, it is very important to im-prove immunity to prevent infectious diseases. A vari-ety of factors such as vaccination failure, infection by immune-suppressive diseases, and abuse of antibiotics can induce immunodeficiency. Use of immune stimula-tors is one solution to improve immunity and to de-crease susceptibility to infectious disease. Herbs that are rich in flavonoids such as thyme extend the activity
Table 6. Effects of dietary thymol + carvacrol supplement on fatty acid composition (%) of serum, thigh, and breast muscles in broilers at 42 d of age
Fatty acid composition1 Control
Thymol + carvacrol (mg/kg)
SEM
Probability
Treatment
Dose response2
60 100 200 Lin. Quad.
Serum ΣSFA 41.1 38.6 34.2 33.5 0.856 0.001 0.002 NS3
ΣMUFA 19.2 20.9 21.2 21.8 1.014 NS NS NS ΣPUFA 39.7 40.5 44.6 44.6 0.540 0.003 0.005 NS n-3 1.30 1.38 1.41 1.53 0.234 NS NS NS n-6 38.4 39.1 43.2 43.6 0.483 0.001 0.004 NSThigh muscle ΣSFA 35.2 32.8 26.9 27.0 0.606 0.004 0.002 0.012 ΣMUFA 28.2 30.6 30.8 31.0 0.560 0.010 0.005 0.027 ΣPUFA 36.7 36.6 42.3 42.0 0.476 0.001 0.016 NS n-3 2.71 2.51 2.80 2.82 0.206 NS NS NS n-6 33.9 34.1 39.4 39.2 0.589 0.001 0.017 NSBreast muscle ΣSFA 33.4 31.8 32.2 31.7 0.595 NS NS NS ΣMUFA 24.5 25.4 25.5 25.4 0.590 NS NS NS ΣPUFA 42.2 42.8 42.3 42.9 0.378 NS NS NS n-3 3.64 3.71 3.70 3.89 0.135 NS NS NS n-6 38.6 39.1 38.6 39.0 0.354 NS NS NS
1ΣSFA: total saturated fatty acids; ΣMUFA: total monounsaturated fatty acids; ΣPUFA: total polyunsaturated fatty acids.2Linear and quadratic effects.3NS, P > 0.05.
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of vitamin C, act as antioxidants, and may therefore enhance immune functions (Acamovic and Brooker, 2005). Because thymol and carvacrol have been report-ed to have antibacterial, antiviral, and antioxidant ac-tivities, an increase in immune responses of chicks is an-ticipated (Botsoglou et al., 2002). Also, Acamovic and Brooker (2005) reported immunostimulating activity of polyphenol fraction of thymol and oregano essential oil with respect to the system of mononuclear phagocyte system, cellular, and humoral immunity.
The effects of dietary thymol + carvacrol on hema-tological parameters and relative weights (g/100 g of BW) of immune organs in broilers at 40 d of age are
shown in Table 9. Heterophil to lymphocyte ratio was linearly reduced in birds fed thymol + carvacrol, but other parameters tested, including white blood cell count, red blood cell count, hemoglobin concentration, and hematocrit percentage, were not influenced by the experimental diets and dose of treatment. Hemato-logical parameters are usually related to health status. These parameters are good indicators of physiological, pathological, and nutritional status of an animal and have the potential of being used to elucidate the impact of nutritional factors and additives supplied in diet. For example, leucocytes are known to increase sharply when infection occurs because they are one of the first
Table 7. Effects of dietary thymol + carvacrol supplement on intestinal and pancreatic digestive enzyme activities (U/mg of digesta protein) in broilers at 24 and 42 d of age
Enzyme activity Control
Thymol + carvacrol (mg/kg)
SEM
Probability
Treatment
Dose response1
60 100 200 Lin. Quad.
Intestine 24 d Trypsin 44.2 46.7 46.8 52.2 0.364 0.001 0.039 NS2
Lipase 24.7 30.2 32.9 34.5 1.927 0.012 0.020 NS Amylase 28.3 27.8 28.1 28.5 3.102 NS NS NS Protease 73.3 89.3 90.8 91.7 3.075 0.020 0.001 0.010 42 d Trypsin 35.2 35.7 35.8 36.6 0.429 NS NS NS Lipase 18.8 19.3 19.4 20.3 0.447 NS NS NS Amylase 13.0 13.4 13.5 13.6 0.470 NS NS NS Protease 73.3 74.4 74.5 74.7 1.104 NS NS NSPancreas 24 d Trypsin 60.4 67.4 73.3 73.5 2.234 0.020 0.004 0.039 Lipase 53.5 54.4 54.5 55.2 0.375 0.037 NS NS Amylase 41.8 42.6 45.5 46.2 2.025 NS NS NS Protease 149 163 167 171 3.860 0.009 0.013 NS 42 d Trypsin 55.3 56.6 57.5 59.3 1.520 NS NS NS Lipase 41.1 41.4 40.9 42.3 0.486 NS NS NS Amylase 34.4 34.1 34.1 35.4 0.523 NS NS NS Protease 148 148 150 150 0.884 NS NS NS
1Linear and quadratic effects.2NS, P > 0.05.
Table 8. Effects of dietary thymol + carvacrol supplement on hypersensitivity (mm) and antibody production (log2) in broilers
Immune response Control
Thymol + carvacrol (mg/kg)
SEM
Probability
Treatment
Dose response1
60 100 200 Lin. Quad.
Hypersensitivity (mm), d 10 24 h after 0.87 0.95 1.13 1.20 0.014 0.001 0.001 NS2
48 h after 0.81 0.86 0.99 1.13 0.031 0.001 0.008 NSSRBC3 injection, d 28 7 d after injection Total anti-SRBC 4.99 5.22 5.35 5.75 0.042 0.001 0.002 NS IgG 3.72 4.01 4.14 4.54 0.035 0.001 0.001 NS 14 d after injection Total anti-SRBC 3.01 3.33 3.47 3.47 0.107 0.015 0.015 NS IgG 2.32 2.68 2.91 2.90 0.119 0.010 0.007 0.045
1Linear and quadratic effects. 2NS, P > 0.05.3SRBC: sheep red blood cell.
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lines of defense in the body (Ganong, 1999). The reli-ability of H/L as a biological index of stress in birds is well documented. The lower H/L observed in broilers fed diets containing the phytogenic product implies the positive influence of thymol + carvacrol on reducing stress in broilers (Bedanova et al., 2007). Reports on the effect of thymol + carvacrol supplementation on blood hematological parameters are very scarce. Unlike our observation, Al-Kassie (2009) showed that diets supplemented with oil extract derived from thyme and cinnamon significantly increased red blood cell, hema-tocrit, hemoglobin, and white blood cell values in broil-ers compared with the control group.
The relative weights of lymphoid organs were not af-fected in birds fed diets with the phytogenic product. In agreement, Rahimi et al. (2011) showed no significant differences in the relative weight of the spleen and bur-sa in broilers fed diet containing thyme compared with control groups. Excessive growth of these lymphoid or-gans may indicate an infection and more mortality in birds.
Phytogenic additives may have more than one mode of action, including affecting feed intake and flavor, stimulating the secretion of digestive enzymes, and increasing gastric and intestinal motility, endocrine stimulation, antimicrobial activity, antiviral activity, anthelminthic activity, coccidiostat activity, immune stimulation, antiinflammatory and antioxidative activ-ity, and pigments. Antimicrobial and antioxidative ef-ficacy of essential oils or the active component of plant extracts have been shown in many in vitro or in vivo studies, but there are still some unanswered questions concerning the mode of action, metabolic pathway, and optimal dosage of phytogenic additives in poultry (Basmacioğlu Malayoğlu et al., 2010).
In conclusion, thymol + carvacrol enhanced BW gain and feed efficiency, and reduced feed intake. Also, the additive increased antioxidant and digestive enzyme activities and improved immune response, which may
beneficially affect health and performance of broiler chickens.
ACKNOWLEDGMENTSThe authors are grateful to the office of the vice
president in research at Ferdowsi University of Mash-had, Iran, for providing the experimental facilities and financial support for this experiment.
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Item Control
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SEM
Probability
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Dose response1
60 100 200 Lin. Quad.
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