Comp Clin Pathol (2011) 20:453–460 DOI 10.1007/s00580-010-1018-1 ORIGINAL ARTICLE Serum biochemical profile and performance of broiler chickens fed diets containing essential oils and pepper Carolina Kist Traesel & Patricia Wolkmer & Candice Schmidt & Cássia B. Silva & Francine C. Paim & Alexandre P. Rosa & Sydney H. Alves & Janio M. Santurio & Sonia T. A. Lopes Received: 16 January 2010 / Accepted: 28 April 2010 / Published online: 30 May 2010 # Springer-Verlag London Limited 2010 Abstract Serum biochemical parameters and performance data were evaluated in broilers fed diets supplemented with antibiotics or essential oils from oregano, sage, rosemary, and pepper crude extract (OLES). Animals (n=910) were distributed into five treatment groups, with seven replicates of 26 birds in each group: the control group (diet without additives); the group receiving an antibiotic growth proC. K. Traesel : C. Schmidt : C. B. Silva : F. C. Paim Postgraduate Program in Veterinary Medicine, Federal University of Santa Maria, Santa Maria, Brazil P. Wolkmer Postgraduate Program in Biochemistry and Toxicology, Federal University of Santa Maria, Santa Maria, Brazil A. P. Rosa Laboratory of Poultry, Animal Husbandry Department, Federal University of Santa Maria, Santa Maria, Brazil S. H. Alves : J. M. Santurio Laboratory of Mycological Research, Department of Microbiology and Parasitology, Federal University of Santa Maria, Santa Maria, Brazil S. T. A. Lopes Laboratory of Veterinary Clinical Analysis, Department of Small Animal Clinical Sciences, Federal University of Santa Maria, Santa Maria, Brazil C. K. Traesel (*) Setor de Virologia, prédio 20, sala 4200, Universidade Federal de Santa Maria, Av. Roraima, nº 1000, Camobi, 97105-900 Santa Maria, RS, Brasil e-mail: ninak13l@yahoo.com.br moter diet; and the groups T50, T100, and T150 (feed supplemented with 50, 100, and 150 mg/kg of OLES, respectively). After 42 days, 55 animals were randomly selected for serum biochemical profile analysis involving pancreatic, renal, and hepatic functions (lipase, amylase, urea, uric acid, aspartate aminotransferase, gamma glutamyltransferase, total cholesterol, high-density lipoprotein, triglycerides, total protein, albumin, globulins, and albumin/globulins ratio). Growth performance of broilers (body weight, weight gain, feed intake, alimentary conversion, and mortality) was also evaluated throughout the experiment period. The increase in serum levels of lipase, uric acid, urea, and aspartate aminotransferase suggests that OLES may cause kidney and liver impairment, mainly, at the higher dose. The OLES dose of 100 mg/kg is suitable for a final body weight and a weight gain similar to those observed in broilers supplemented with antibiotic growth promoters. Keywords Kidney . Liver . Plants extract . Poultry . Growth performance Introduction Growth promoters based on antibiotics have been used in animal diets to improve animal performance (Butaye et al. 2003). The use of these growth promoters has been restricted due to the possibility of selecting antibioticresistant microorganisms, the development of bacterial resistance in humans, and the growing demand for antibiotic residue-free food products (Butaye et al. 2003; Saleha et al. 2009). Alternatively, the use of essential oils that possess antimicrobial potential has been studied (Lee et al. 2004a; Santurio et al. 2007). 454 Essential oils are composed of a complex mixture of active substances extracted from plants through a steam distillation process or generated via chemical synthesis (Lee et al. 2004a; Zhang et al. 2005). The concentration of the biologically active components in essential oils is variable and dependent on the species, the part of the plant used, soil, environmental conditions, and time of harvest (Kamel 2000; Lee et al. 2004a). These substances can produce several beneficial effects, such as an increase in animal performance (Hernández et al. 2004; Zhang et al. 2005; Kadam et al. 2009). Oregano (Kamel 2000), sage (Tzakou et al. 2001), and rosemary (Farag et al. 1989) extracts present antimicrobial activity against different gram (±) bacteria and other microorganisms, and red pepper has antidiarrheal and anti-inflammatory properties (Kamel 2000). When fed in combination, the extracts were more effective than the individual components (Kamel 2000; Burt 2004). However, essential oils can also produce toxic effects in chickens when used in high doses (Lee et al. 2004a). The response to essential oils may be dose dependent (Zhang et al. 2005). The effects of essential oils in the clinical chemistry of broilers are still unclear. Serum concentrations of amylase and lipase in broilers can be measured for pancreatic function evaluation. An increase in these serum parameters might be related to a pancreatic (Lumeij 1997) or renal injury (González and Silva 2006). Avian renal function can be evaluated by serum urea and uric acid measurements, the latter being a more reliable parameter; the elevation of these parameters in the serum occurs when 30% or less of the kidneys are functional (Lumeij 1997; Campbell 2007). In birds, an increase in serum urea levels occurs after a decrease in glomerular filtration rate and may indicate a kidney disease or a physiological response to fluid restriction. However, uric acid excretion occurs via tubular secretion, which is slightly influenced by urine flow and hydration state, increasing only when there are very severe prerenal causes and extensive tubular damage (Lumeij 1997; Phalen 2000). Serum parameters can also be elevated after high protein intake, since they are involved in nitrogen metabolism (Campbell 2007; Schmidt et al. 2007). Hepatic function of birds can be evaluated by serum concentration of liver enzymes aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT), cholesterol, and albumin, since its synthesis occurs in the liver (González and Silva 2006). Increase AST serum levels may be caused by hepatocellular disease (Campbell 2007), while GGT elevation is usually related to hepatobiliary disease (Tennant 1997). However, AST is not a specific liver injury enzyme and may also be altered by muscle injuries, as indicated by a concurrent increase in creatine kinase (CK) levels (Tennant 1997; Campbell 2007; Schmidt et al. 2007). Therefore, the aim of this study was to evaluate Comp Clin Pathol (2011) 20:453–460 the effect of different doses of oregano, sage, rosemary essential oils, and chili pepper crude extract as dietary supplements on pancreatic, renal, and hepatic functions of broilers by analyzing their serum biochemical profile and on broiler performance. Materials and methods Animals and location One-day-old male Cobb chicks (n=910) with an average weight of 42 g were housed at the appropriate temperature according to the age, remaining under natural light cycles, at 33°C during the first 7 days of life and gradually decreased to room temperature (24–25.5°C) after this period of time. The animals were distributed in 35 boxes (7 boxes per group) of 26 birds each, containing wood shaving litter and allowed with free access to food (the composition of the basal diet is described in Table 1) and drinking water. Chickens were reared in boxes until 42 days of age. Essential oils and crude extract Essential oils derived from oregano (Origanum vulgare L.), sage (Salvia officinalis L.), rosemary (Rosmarinus officinalis L.), and chili pepper (Capsicum frutescens L.) crude extract (OLES) were produced by the Mycological Research Laboratory according to Pozzatti et al. (2008). The major components of mixed essential oils (chemical composition analyzed by gas chromatography and mass spectrometry) were as follows: carvacrol (oregano 92.6%), camphor (sage 13.9% and rosemary 26.31%), cis- and trans-thujone (sage 55.71%), 1,8-cineole (sage 7.54% and rosemary 28.59%), α- and β-pinene (sage 5.92% and rosemary 24.15%), and camphene (sage 2.51% and rosemary 11.76%). Chili pepper crude extract obtainment was carried out following the literature (Li et al. 2009): dried and powdered C. frutescens was extracted three times with 60% ethanol at 60°C; the ethanol solutions were combined and evaporated by rotary vaporization at 60°C under reduced pressure. OLES were microencapsulated due to their volatile properties and also to homogenate within the ration. Experimental groups and feeding The birds were randomly allocated to receive one of five diets (seven replications of 26 birds per treatment group). Animals from the control treatment group (Tc) received a diet based on corn, soy bean meal, and microelements necessary for the animal’s maintenance (Table 1). The second treatment group (Tatb) was fed with the same diet Comp Clin Pathol (2011) 20:453–460 455 Table 1 Composition and nutritional profile of the basal diet during specific periods of age All five treatment groups received the basal diet. broilers supplemented with antibiotics (Tatb) received vitamin premix (0.50%) with antibiotic grow promoters (AGP). Essential oils of oregano, sage, rosemary, and chili pepper crude extract were added in the proportion of 0.005%, 0.010%, and 0.015% in T50, T100, and T150, respectively. The composition was completed (100%) with the addiction of kaolin within the ration (0.100% in the control group and Tatb; 0.095%, 0.090%, and 0.085% in T50, T100, and T150 respectively) CP crude protein Ingredients (%) Corn Soybean meal (45.27% CP) Vegetable oil Dicalcium phosphate Calcium carbonate Salt (NaCl) Vitamin premix without AGP L-lysine DL-methionine Nutrients CP (%) Metabolizable energy (kcal/kg) Calcium (%) Available phosphorus (%) Lysine (%) Total sulfur amino acids (%) Threonine (%) Tryptophan (%) as the Tc group, but included supplements containing antibiotic growth promoters, at the dose suggested by the manufacturer (colistin sulfate+oxytetracycline 0.005%; Table 1). In treatment groups T 50, T 100, and T150, encapsulated OLES were added to the antibiotic-free feed at proportions of 50, 100, and 150 mg/kg, respectively. Collection of blood samples This study was approved by the Committee on Ethics and Animal Welfare of the Rural Science Center of the Federal University of Santa Maria (CCR/UFSM, no. 23081. 014222/2007-37) in accordance with existing legislation and ethical principles, as published by the Brazilian College of Animal Experiments (COBEA). At 42 days of age, 14 animals were randomly selected from each group (two birds of each box, a total of 70 broiler chickens in all five treatments) and slaughtered after electrical stunning. Approximately 6 ml of blood was collected by cardiac puncture and stored in tubes without anticoagulant. Serum was obtained by centrifugation and stored at −20°C for further serum biochemical profile analysis. Biochemical assay Only 11 sera samples out of the 14 obtained per group were suitable to perform all the serum biochemical measurements, mainly because of hemolysis and/or volume of the 1–7 days 8–21 days 22–35 days 36–42 days 56.54 36.85 2.57 1.81 1.04 0.40 0.50 0.08 0.11 57.70 35.64 2.81 1.76 0.95 0.40 0.50 0.06 0.08 59.70 33.21 3.26 1.78 0.96 0.40 0.50 0.04 0.06 61.99 30.80 3.62 1.69 0.90 0.40 0.50 0.00 0.00 22.00 2970 1.00 0.45 21.00 3000 0.95 0.44 20.50 3050 0.95 0.44 19.50 3100 0.90 0.42 1.30 0.92 0.84 0.23 1.25 0.88 0.82 0.22 1.16 0.83 0.79 0.21 1.06 0.75 0.75 0.20 sample. Serum lipase concentration was measured using a commercial kit (Human do Brazil, Núcleo Diagnóstico Produtos Especializados Ltda.- Itabira - MG - Brazil), and the absorbance was evaluated by spectrophotometry at 412 nm. The levels of serum amylase, urea, uric acid, AST, CK, GGT, total cholesterol, high-density lipoprotein (HDLcholesterol), triglycerides, total protein (TP), and albumin measurements were done in a semiautomatic analyzer (TP Analyzer Plus; Thermoplate - China), using commercial kits (Labtest Diagnóstica S.A.- Lagoa Santa - MG - Brazil). All tests were carried out in duplicates. Globulins values were obtained from the difference between serum TP and serum albumin concentrations; the albumin/globulins ratio was also calculated. Performance data Live body weight of broilers was recorded on days 1, 7, 21, 35, and 42; birds were group weighed by pen. The body weight gain and the feed consumption were determined at each feed change interval; they were calculated by means of the difference between the final and the initial broilers weight and amount of ingested food, respectively, in the periods between days 1 and 7, 8–21, 22–35, and 36–42 and also from days 1 to 42 (total experiment period). Data for feed conversion (feed intake/weight gain) and mortality rate estimation were also obtained at the same periods. Birds were checked for mortality twice daily. 456 Statistical analysis Each treatment effect of biochemical assays was evaluated by analyzing the factor variance using PROC GLM (General Linear Models Procedure); when a treatment effect was found on the dependent variable, the difference between each group was examined by the least squares means test. The performance data were analyzed with Tukey’s test when appropriate. All dependent variables were tested for normality using the ShapiroWilk Test, and if necessary, they were normalized according to data distribution. The analyses were performed using the statistical package, SAS (SAS Institute, Cary, NC), with a significance level of 5% (P<0.05). The values are reported as the mean ± standard error. Results The serum lipase concentration was higher in animals that received OLES in groups T50, T150 (P<0.05), and T100 (P<0.01) than in the control animals (Tc; Fig. 1a). There was no significant difference in serum amylase levels. The serum levels of urea were higher than control in the groups T50 (P<0.01) and T150 (P<0.05; Fig. 1b). The serum levels of uric acid increased gradually from groups T50 to T150. The uric acid showed a significant increase in groups T50 (P<0.01), T100 (P<0.05), and T150 (P<0.001) compared with Tc. Moreover, the values of uric acid in T150 were significantly higher than in animals treated with antibiotics (Fig. 1c). Fig. 1 Mean values and standard errors of serum lipase (a), urea (b), uric acid (c), and AST (d) of 42-day-old broilers fed basal diet (Tc) supplemented with antibiotics (Tatb) or with essential oils of oregano, sage, rosemary, and chili pepper crude extract in the proportion of 50 (T50), 100 (T100), and 150 (T150) mg/kg. * Represents statistical difference (P< 0.05) when compared with Tc; † indicates a significant difference (P<0.05) when compared with Tatb; ‡ represents a statistical difference (P<0.05) when compared with T50. The difference between each group (n=11) was examined by the least squares means test, using the statistical package SAS. Attempt to different graph scales Comp Clin Pathol (2011) 20:453–460 The values of AST were higher in T150 than in the Tc (P<0.01), Tatb, and T50 (P<0.05) groups. The average measurements of AST were also higher in the T100 group than in Tc (P<0.05; Fig. 1d). There was no significant difference among groups in serum levels of CK and GGT. Total serum cholesterol, HDL-cholesterol, and triglycerides showed no significant difference among groups. Also, no significant difference was detected in serum TP, albumin, globulins, and albumin/globulins ratio in the biochemical analysis. In Table 2, the nonsignificant biochemical results are presented. The live body weight of 42-day-old broilers (Fig. 2) and body weight gain from 1 to 42 days (Table 3) were similar between Tatb and T100. The means were higher in these groups than in the control group (P < 0.001). No significant differences in average feed consumption, feed conversion rate and mortality percentage were observed among the treatment groups between days 1 and 42 (Table 3). Discussion OLES were derived from oregano, sage, rosemary essential oils, and chili pepper crude extract. The major active component of mixed essential oils was carvacrol, and the active constituents of pepper are capsaicenoids, or capsaicin (Carvalho et al. 2005). Components were selected by their individual antibiotic activity (Farag et al. 1989; Kamel 2000; Tzakou et al. 2001) and disponibility, constituting a viable alternative to growth promoters. A synergistic effect (Burt 2004; Zhang et al. 2005) was sought when the Comp Clin Pathol (2011) 20:453–460 457 Table 2 Serum biochemical parameters of 42-day-old broilers fed basal diet (Tc), supplemented with antibiotics (Tatb), and with essential oils of oregano, sage, rosemary, and chili pepper crude extract in the proportion of 50 (T50), 100 (T100), and 150 mg/kg (T150) Amylase (U/l) CK (U/l) GGT (U/l) Total cholesterol (mg/dl) HDL-cholesterol (mg/dl) Triglycerides (mg/dl) TP (g/dl) Albumin (g/dl) Globulins (g/dl) A/G Tc Tatb T50 T100 T150 1,133.74±96.30 4,998.04±537.48 22.46±4.03 104.56±4.01 61.47±3.16 39.81±1.82 3.36±0.11 1.57±0.04 1.80±0.11 0.90±0.06 1,126.28±124.83 5,371.12±801.41 24.40±3.55 115.01±4.40 71.28±4.45 32.75±3.38 3.42±0.07 1.56±0.05 1.86±0.08 0.86±0.06 1,150.68±216.17 5,445.39±691.98 22.96±4.43 111.50±5.03 70.20±3.84 33.05±2.35 3.70±0.18 1.57±0.10 2.12±0.13 0.77±0.06 920.22±90.53 5,293.65±1034.98 27.12±2.14 114.97±5.15 73.02±3.47 30.35±3.14 3.36±0.13 1.56±0.07 1.79±0.11 0.91±0.07 1,350.33±294.15 5,227.71±927.27 22.89±1.65 118.23±8.60 64.25±4.12 29.37±2.74 3.53±0.33 1.52±0.06 2.00±0.36 0.98±0.17 The results are represented as mean ± standard error, n=11. The difference of each group was examined by the least squares means test, using the statistical package SAS, and no significant difference was found (P>0.05) CK creatine kinase, GGT gamma glutamyltransferase, HDL high-density lipoprotein, TP total protein, A/G albumin/globulins ratio combination of essential oils and/or their active compounds was used together. In the present study, the serum concentrations of amylase and lipase in broilers fed OLES were measured for pancreatic function evaluation. Although there are beneficial effects of essential oils on digestibility (Hernández et al. 2004) through the stimulation of pancreatic enzymes (Jang et al. 2007), an increase in these parameters in the serum might be related to a pancreatic injury, as acute pancreatitis or pancreatic necrosis (Lumeij 1997), which lead to secretion of enzymes into blood. Additionally, the changes of enzyme activity hyperamylasemia and hyperlipasemia can also occur in renal injury, when their excretion is reduced by a decrease in glomerular filtration (González and Silva 2006). In animals from groups T50, T100, and T150, the increase in serum levels of lipase is probably related to a renal injury, which is reinforced by the results obtained by the renal function evaluation. The observed increases in both serum uric acid and urea in this study were probably due to an initial kidney function impairment caused by OLES, since the animals were fed a balanced diet with crude protein levels according to the birds’ age (Table 1) and were allowed free access to food and drinking water throughout treatment period. Besides, there was no significant alteration in feed intake or in serum TP levels in broilers fed OLES, as demonstrated in Table 3 and 2, respectively. The level of kidney damage seems to correlate with the OLES dose, because the levels of uric acid gradually elevated with an increase in the OLES dose, suggesting a dosedependent effect (Fig. 1c). This early kidney function impairment is not clinically apparent. There was no statistical evidence of a reduction in growth performance and mortality with increasing levels of OLES, probably due to the short exposition period. Conversely, Ghazalah and Ali (2008) found reduced serum uric acid levels in broilers fed diet with 0.5% dried rosemary leaf meal. Essential oils can produce toxic effects in chickens when administered in high doses; however, more studies are needed to define safety levels in broilers; in rats, the average lethal dose (LD50) of carvacrol—the major component of OLES—is about six times higher than the highest dose used in this experiment (Lee et al. 2004a). In our study, the decrease in urea and uric acid excretion associated with kidney disease in birds (Lumeij 1997) could be related to OLES toxicity. Besides, the active compounds are excreted in the urine (Kohlert et al. 2000). Sage and rosemary at high doses and the prolonged use could cause renal failure and nephritis, respectively (Plantamed 2009; Canto Verde 2010). It would appear that Fig. 2 Average live body weight of 42-day-old broilers fed basal diet (Tc) supplemented with antibiotics (Tatb) or with essential oils of oregano, sage, rosemary, and chili pepper crude extract in the proportion of 50 (T50), 100 (T100), and 150 (T150) mg/kg. a>b (P< 0.001), the values for the parameters are significantly different when the letters are different; the difference between each group was examined with Tukey’s test 458 Comp Clin Pathol (2011) 20:453–460 Table 3 Effect of essential oils and pepper extract on performance of broilers (1–42 days) Treatments Body weight gain (g) Feed consumption (g) Feed conversion / weight gain (g) Mortality (%) Tc Tatb T50 T100 T150 Mean 2,495.74±20.71b 2,596.55±11.66a 2,550.45±13.58ab 2,594.13±18.30a 2,560.41±16.64ab 2,559.46 4,731.78±64.41 4,820.97±43.97 4,734.99±37.93 4,815.49±63.04 4,839.32±80.47 4,788.51 1.90±0.02 1.86±0.02 1.86±0.01 1.86±0.02 1.89±0.02 1.87 3.30±1.77 2.75±1.38 1.10±0.71 2.20±1.14 6.04±1.85 3.08 The results are represented as mean ± standard error. The difference of each group was examined with Tukey’s test; a>b, the values for the parameters are significantly different (P<0.05) when the letters are different; the absence of letters represents no significant difference Tc control group (basal diet), Tatb broilers supplemented with antibiotics, T50, T100, and T150 broilers supplemented with essential oils of oregano, sage, rosemary, and chili pepper crude extract in the proportion of 50, 100, and 150 mg/kg, respectively sage oil is the compound most likely to be causing the possible kidney and liver-associated effects due to its content of a toxic ketone known as thujone (Lima et al. 2004; Craig 2007). Sage essential oil in this study presented 55.71% of cis- and trans-thujone. Although thujone has been identified as a toxic agent, it is still unclear whether it is toxic to birds. Interestingly, the serum levels of uric acid and the liver enzyme, AST, in the group T150 were significantly higher than in the group Tatb (besides in the Tc); this is an important finding because the supplementation with antibiotics growth promoters is the most common approach in poultry’s routine. Serum concentration of AST also increased concomitantly with OLES doses, suggesting a dose-dependent effect (Fig. 1d). The increase in serum levels of AST is caused by hepatocyte injury, resulting from necrosis or changes in cell membrane permeability and can be attributed to recent liver dysfunction (Tennant 1997). In our study, there was no significant increase in serum levels of CK and no evidence of muscle injury; therefore, it is more likely that the increased AST levels (in OLES doses greater than 100 mg/kg) originated in the liver and not the muscle, since elevation in plasma AST activities without an increase in CK levels suggests hepatocellular disease (Campbell 2007). Besides, essential oils are quickly metabolized in the liver (Hood et al. 1978; Kohlert et al. 2000), and this can overload the liver causing damage, suggesting that the increased serum AST observed was due to an initial hepatic injury. Ghazalah and Ali (2008) also observed low serum AST levels in control groups compared with groups of broilers fed 0.5% of dried rosemary leaf meal in the diet. Potentially, toxic effects of sage essential oil especially to the liver, the main detoxifying organ, were reported by Lima et al. (2004); at doses higher than 200 nl/ ml acting directly on freshly isolated rat hepatocytes, the essential oil may cause toxicity. This hepatotoxicity as well as the neurotoxicity of thujones and camphor—major compounds of S. officinalis essential oil—justifies concerns with the consumption of high doses of sage products. No significant difference in GGT serum concentration was observed among treatment groups, suggesting that biliar cholestasis and duct hyperplasia (Tennant 1997) did not occur in this experiment. Moreover, serum GGT elevation in birds with hepatobiliary disease is not predictable, which may or may not occur, depending on the nature of the hepatic injury and the species of bird (Campbell 2007; Schmidt et al. 2007). Total serum cholesterol, HDL-cholesterol, and triglycerides levels showed no significant differences between groups. These results were similar to those found by Lee et al. (2003, 2004b), and Bampidis et al. (2005) in studies with broiler chickens and turkeys using different active compounds (carvacrol, cinnamaldehyde, thymol) and essential oils (oregano and CRINA Poultry). However, several active compounds such as thymol, carvacrol, and borneol can cause hypocholesterolemia by inhibiting the regulatory enzyme of cholesterol synthesis, 3-hydroxy-3methylglutaryl coenzyme A reductase (Case et al. 1995; Lee et al. 2004a), evidencing that it could have different effects on triglyceride metabolism in broiler chickens. No significant differences were observed between groups in the serum levels of TP, albumin, globulins, and albumin/globulins ratio. The obtained results, as normal serum albumin levels and albumin/globulins ratio, exclude the presence of hepatic insufficiency in our study (but not exclude hepatic disease), since albumin synthesis occurs in the liver (González and Silva 2006). Similar results were found in a study by Abd El-Hakim et al. (2009) with broilers fed herbs and/or organic acid. These authors also suggested that the effects of plant extracts on plasma proteins are species-specific. The results obtained by Ghazalah and Ali (2008) showed significant increases in total protein and globulins fractions serum values when broilers were fed diets with 0.5% rosemary dried leaf meal. Comp Clin Pathol (2011) 20:453–460 Additionally, the evident antibacterial activity (Farag et al. 1989; Kamel 2000; Tzakou et al. 2001), the improvement in digestibility (Hernández et al. 2004) and in feed utilization (Zhang et al. 2005; Kadam et al. 2009), and the digestive and pancreatic enzymes stimulation (Lee et al. 2003; Jang et al. 2007) in response to essential oil ingestion might increase animal performance (Hernández et al. 2004; Zhang et al. 2005; Kadam et al. 2009). Our study also demonstrated that the treatment with 100 mg/kg of OLES correlates with higher live body weights at 42 days and weight gain from 1 to 42 days in broilers, in comparison with the control group. Moreover, these results were similar to that observed in the antibiotics growth promoters group, which are currently and frequently employed in poultry’s routine. Due to their real importance for this study and because the results of all periods were too extensive, the only data presented here are final body weight and performance data from days 1 to 42 (Fig. 2; Table 3). Hernández et al. (2004) observed that the effect of different additives containing oregano and pepper essential oils (200 mg/kg) or sage and rosemary extracts (5,000 mg/kg) on digestibility improved the broiler performance slightly. Botsoglou et al. (2002) indicated that dietary oregano oil exerted no growth promoting effect on broilers when administered at 50 or 100 mg/kg of feed and, oregano or red pepper extracts individually added to broiler feeds at 200 mg/kg did not influence broiler performance (Barreto et al. 2008). However, when OLES were used together, the dose of 100 mg/kg was suitable. Conclusions Our findings suggest that OLES may cause renal and hepatic functions impairment at levels as high as 150 mg/ kg. However, suitable broilers’ final live body weight and body weight gain are obtained at OLES proportion of 100 mg/kg, constituting the ideal dose. More studies are necessary using individual plants extracts and mixtures to optimize the essential oil combinations that will provide benefits to the animal without being harmful. Moreover, the effect of over time and continuous OLES supplementation on pancreatic, renal, and hepatic functions of birds that live more than 42 days and other animal with longer lives should be investigated, because further damage can occur with frequent and repeated exposure. Acknowledgements This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, nº 476507/ 2007-3) and Ministério da Ciência e Tecnologia (MCT/CNPq, 15/ 2007-Universal). 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