Effects of curcumin or nanocurcumin on blood biochemical parameters, intestinal morphology and microbial population of broiler chickens reared under normal and cold stress conditions

ABSTRACT This study was conducted to evaluate the effect of curcumin/nanocurcumin on blood parameters, intestinal morphology and microbial population in broiler chickens reared under normal and cold stress conditions. The experiment was designed with two identical houses; each consisted of five diets with 5 replicates of 10 birds each. The diets were (1) control; (2) and (3) Control + 200 or 400 mg/kg curcumin; (4) and (5) Control + 200 or 400 mg/kg nanocurcumin, respectively. Birds in both houses were reared under commercial temperatures until day 14. The temperature in the first house was maintained according to the commercial practices, whereas the temperature in the second house dropped to 15°C on day 14 and maintained between 13–15°C until day 42. Total weight gain was decreased, but plasma malondialdehyde (MDA), liver enzymes activities and heterophils/lymphocytes ratio were increased in cold-stressed birds compared to those that grew in normal temperature. Supplementation of curcumin/nanocurcumin in diet improved the weight gain and villus surface area of birds in concomitance to lower their plasma MDA, liver enzymes activities, caecal E. coli population compared to those fed control diet. It is concluded that the addition of 200 mg/kg curcumin/nanocurcumin to diet may improve the performance, antioxidant status and jejunal tissue health in broiler chickens.


Introduction
Birds may suffer a variety of environmental stress such as cold stress, which affect animal health and welfare (Tsutsayeva & Sevryukova 2001). Cold stress is related to disease and tissue damage (Dhanalakshmi et al. 2007). Thermogenic mechanisms are based on the increased secretion of thyroid hormone and increased metabolic rate in birds (Guo et al. 2007). Increased metabolic rates lead to increased tissue requirements for oxygen (Currie 1999). An imbalance between oxygen demand and oxygen supply results in hypoxaemia (Hassanzadeh 2010). Hypoxia may increase the production of free radicals (Bottje & Wideman 1995). The increase in the aspartate aminotransferase and lactate dehydrogenase (AST/LDH) activities is an indicator of advanced liver cells damage followed by the increased production of free radicals (Arab et al. 2006). Antioxidants have a major role in protecting cells from the actions of reactive oxygen species (ROS) by reducing chemical radicals and disrupting the process of lipid peroxidation. Lipid peroxidation is an important feature of cellular damage due to free radicals attack (Ahmad et al. 2012). Using synthetic antioxidants, butylated hydroxyl anisole (BHA) and butylated hydroxyl toluene (BHT), have been restricted long ago, because of their possible carcinogenicity causing liver swelling and changing liver enzyme activities (Martin & Gilbert 1968). Recently, curcumin, the yellow pigment of turmeric, is considered. Curcumin is a compound that has a wide range of therapeutic activities including antioxidant (Sandur et al. 2007), free radical scavenging (Zhang et al. 2014), protection of biological membranes against peroxidative damage (Priyadarsini 1997), increased immuno function (Cleary 2004), antiviral and antibacterial (Singh et al. 2010) and hypolipidemic effect in rat (Rao et al. 1970). Poor bioavailability of curcumin due to low absorption, fast metabolism and fast systemic elimination from the body (Anand et al. 2007) has been a major issue. One of the suggestions to improve the bioavailability of curcumin is the use of curcumin nanoparticles which increases the oral absorption of curcumin (Nabavi et al. 2014). It has been demonstrated that curcumin in the form of nanoparticles (micellar curcumin) increased the in vivo bioavailability and tissue distribution and gave a 60-fold higher biological half-life (Ma et al. 2007) compared with the native curcumin treatment in a rat model. This study was conducted to compare the effect of curcumin/nanocurcumin on blood biochemical parameters, intestinal morphology and microbial population in broiler chickens reared under normal and cold stress conditions.

Birds, diets and housing
This experiment was conducted as a split plot, using two identical houses, each with 250 day-old Ross 308 male broiler chicks, allocated to 25 pens (mean body weight 43 ± 1.2) with five replicates of 10 birds each and fed five different diets. A mash-based corn-soybean meal diet for starter (1-10), grower (11-24) and finisher (25-42) periods was formulated according to Ross 308 nutrient recommendations (Aviagen 2014; Table 1). Curcumin/ nanocurcumin was also added to the basal diet of each period to prepare five dietary treatments including (I) control (zero level of curcumin/nanocurcumin); (II) 200 mg/kg curcumin; (III) 400 mg/kg curcumin; (IV) 200 mg/kg nanocurcumin and (V) 400 mg/kg nanocurcumin. Diets and water were provided ad libitum throughout the experiment. The lighting programme was 23 h light and 1 h dark from day one to the end of the experiment. The temperature of both houses were set at 32°C for the first week and then reduced to 29°C in the second week. The temperature of one house was gradually decreased by approximately 0.5°C every other day from day 14 until it reached 22°C on 28 days of age and remained constant thereafter. The temperature in the second house gradually dropped to 15°C on day 14 and maintained between 13 and 15°C thereafter. The experimental protocol was reviewed and approved by the Animal Care Committee at the Ferdowsi University of Mashhad.

Curcumin and nanocurcumin
Curcumin was obtained from Sami Labs Limited, Bangalore, India, and used without any treatment. The commercially available source of curcumin is usually composed of 77% curcumin, 18% demethoxycurcumin and 5% bisdemethoxycurcumin (Basnet et al. 2010). Nanocurcumin, used in our experiment, was a nanomicelle containing curcumin and registered as curcumin product (SinaCurcumin ® ) for human oral use which has been developed in the Nanotechnology Research Center of the Mashhad University of Medical Science and marketed by Exir NanoSina Company, Tehran, Iran (IRC:1228225765). Nanocurcumin is prepared from GRAS (generally recognized as safe) pharmaceutical excipients and C3-complex form of curcumin. The encapsulation of curcumin in this nanomicelle is near 100% and the sizes are around 10 nm. Nanocurcumin has a significantly higher bioavailability after oral use compared to curcumin powder (Rahimi et al. 2015).

Performance
The pen weight of birds and feed intake were recorded and the corrected feed conversion ratio (FCR) was calculated from the replicate weight gain and feed intake corrected for mortality for the whole experimental period (1-42 days).

Blood parameters
One bird from each replicate pen was randomly selected at 42 day of age and blood sample was taken from wing vein, into a heparinized syringe. A drop of whole blood from each sample was used to prepare the smears on microscopic slides, dried and fixed with methanol. All smears were stained with May-Grunwald and Giemsa stains (Lucas & Jamroz 1961). In each slide, 100 white blood cells (WBC) were numerated and percent of heterophils (H), lymphocytes (L) and H/L ratio was recorded. Remaining portion of blood was centrifuged and the plasma samples stored at -20°C for later analysis (Tankson et al. 2002). Plasma total cholesterol, (high density lipoprotein (HDL), low density lipoprotein (LDL) cholesterol), triglyceride, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities were measured using commercial diagnostic kits (BioSystems S. A. Barcelona. Spain) by using an autoanalyser (Technicon RA1000, Bayer Diagnostics, Puteaux, France). Plasma malondialdehyde (MDA) was determined based on colorimetric assay of thiobarbituricacid reactive substances as described by Rao et al. (1989).

Intestinal histomorphology
The selected birds were slaughtered after the withdrawal of blood samples at 42 days of age. Samples of jejunum (2-cm segments) were obtained from the mid part of the jejunum and flushed by 0.9% saline, then immersed in a formalin solution (10%) for 72 h and processed according to the method described by Iji et al. (2001) and embedded in paraffin wax. Processed sections were cut (2 μm) using a microtome, placed on a glass slide and then stained with haematoxylin-eosin. The samples were analysed under a light microscope to determine morphometric indices. The morphometric variables including villus height (VH), crypt depth (CD), VH/CD ratio and villus width (VW) were recorded and the villus surface area (VS) was calculated using the following formula: (2π) × (VW/2) × (VH) (Sakamoto et al. 2000). There were three cross-sections per sample and the mean from five villi per cross section was used as the average value for analysis.

Bacterial populations
The same slaughtered birds were also used for the enumeration of microbial population. Caecal contents of each bird was separately collected by gently squeezing into a tube and stored at −80°C for the enumeration of microbial population. The sample tubes were later placed at room temperature until thawed and then 1 g from each sample was homogenized in 9 mL sterile water and serial dilutions were prepared. A volume of 100 μL from the concentration of 10 −3 and 10 −4 , which were determined as suitable density for E. coli and Lactobacilli, respectively, was separately removed and smeared onto plates containing Macconkey agar and Man-Rogosa-Sharpe (MRS) agar medium and completely distributed to all parts of each plate. The number of Lactobacillus on MRS agar and E. coli on Macconkey agar was counted after the plates were incubated at 37°C in an anaerobic chamber for 48 h and in an aerobic chamber for 24 h, respectively (Güban et al. 2006). Bacterial population were reported as logarithmic number of bacteria per 1 g of caecal sample.

Statistical analysis
The analysis of variance was performed using the general linear models procedure of SAS software based on a split plot design (SAS Institute Inc 2004). The significant difference between treatment means was determined by the Tukey test (p < .05). Moreover, orthogonal contrasts were used to compare mean response variables curcumin/nanocurcumin vs control diet and curcumin vs nanocurcumin.

Performance
The interaction effect of temperature and diet curcumin/nanocurcumin was not significant for the feed intake and FCR for the whole experimental period. Feed intake and FCR significantly were higher in cold-stressed birds than in those reared under normal temperature (107.3 vs 102.3 g/b/day and 1.87 vs 1.71, respectively) for the 42-day experimental period. Supplementation of diet with curcumin/nanocurcumin did not affect birds' feed intake, but nanocurcumin at the rate of 200 mg/kg improved (P < .05) the FCR of birds compared to those fed control diet (1.73 vs 1.83) (data not presented).
The interaction effect of temperature and diet was significant on weight gain for the whole experimental period. Supplementation of diet with 400 mg curcumin or 200 mg nanocurcumin per kg in cold-stressed birds significantly improved weight gain compared with that in those fed control diet during the 42-day experimental period (58.1, 59.5 vs 55.7 g/b/day). Cold-stressed birds fed 200 mg/kg nanocurcumin had similar weight gain compared to those fed control diet and reared under normal temperature (59.5 vs 59.7 g/b/day). The weight gain of cold-stressed birds was significantly lower than that of those reared under normal temperature (57.3 vs 59.8 g/b/day).

Malondialdehyde and liver enzymes activities
Malondialdehyde (MDA), LDH and AST activities in birds fed diets supplemented with curcumin/nanocurcumin and reared under normal or cold environmental conditions are shown in Table 2. Plasma MDA, LDH and AST were increased in coldstressed birds compared to that in those reared under normal conditions (p < .05). Using orthogonal contrast, birds fed diets supplemented with curcumin/nanocurcumin had lower plasma LDH activity and MDA than those fed control diet. Plasma MDA in birds was increased when curcumin/nanocurcumin supplementation increased from 200 to 400 mg/kg diet (p < .05). Using the orthogonal contrast, there was not a significant difference between plasma MDA in birds fed diet containing either curcumin or nanocurcumin. But, LDH/AST activities in birds fed diet supplemented with nanocurcumin were significantly higher than that in those fed curcumin diet. Means in the same column for each effect with no common superscript are significantly different.

Blood cholesterol
The effect of curcumin/nanocurcumin supplemented diet on total cholesterol (Tchol), HDL, LDL and triglycerides (TG) in plasma of birds reared in cold-stressed or normal environmental temperature is shown in Table 3. Cold stress increased Tchol, HDL, LDL and TG in the plasma of birds compared with that in those reared in normal environmental conditions (p < .05).
Using orthogonal contrasts, the supplementation of diet with curcumin/nanocurcumin decreased Tchol, LDL and TG in the plasma of birds (p < .05) compared to that in those fed control diet. The amount of Tchol, HDL, LDL and TG in the plasma of birds fed diet containing nanocurcumin was significantly higher than that in those fed curcumin diet. Birds fed curcumin diet and exposed to cold stress had similar LDL compared to those fed control diet and grew under normal environmental temperature.

Immuno function
The effect of dietary curcumin/nanocurcumin supplementation on the number of WBC, H, L and H/L ratio in birds exposed to cold stress or normal temperature conditions is shown in Table 4. Cold stress significantly decreased the number of WBC, L and increased the number of H and H/L ratio compared to that of those reared at normal environmental temperature (p < .05). Using orthogonal contrasts, the supplementation of diet with curcumin/nanocurcumin significantly increased the number of WBC, L and decreased the number of H and H/L ratio in birds compared to that in those fed control diet (p < .05). The number of H and H/L ratio in birds fed diets supplemented with nanocurcumin was significantly higher than that in those fed curcumin-supplemented diet. Birds fed curcumin diet and exposed to cold stress had similar L and H/L ratio compared to those fed control diet and grew under normal environmental temperature.

Intestinal histomorphology
The effect of curcumin/nanocurcumin supplementation of diet on jejunal histomorphology of birds reared in normal or cold environmental conditions is shown in Table 5. Cold stress significantly decreased VH, VW and VS (figure 1). But there was no significant difference between CD or VH/CD in birds reared in normal condition compared to those reared in cold stress. Using orthogonal contrasts, VH, VH/CD and VS in birds fed curcumin/nanocurcumin supplementation were higher than that in those fed control diet. There was no significant difference between jejunal histomorphological parameters in birds fed diet containing either curcumin or nanocurcumin.

Bacterial populations
Caecal microbial population in birds fed diet supplemented with curcumin/nanocurcumin and reared in normal or cold environmental condition is shown in Table 6. Caecal E. coli in cold-stressed birds was higher than that in those reared in normal temperature condition (p < .05). However, the cold stress significantly decreased the caecal lactobacilli count. The Means in the same column for each effect with no common superscript are significantly different. Orthogonal contrast. Means in the same column for each effect with no common superscript are significantly different. Orthogonal contrast. use orthogonal contrast indicated that the number of E. coli and lactobacilli in birds fed curcumin-supplemented diet was significantly lower and higher than that in those fed control diet, respectively. Nanocurcumin supplementation decreased the number of E. coli and increased lactobacilli count as compared to those fed diet containing curcumin (p < .05).

Discussion
Supplementation of diet with curcumin/nanocurcumin increased weight gain in birds compared to those fed control diet and reduced the negative effect of cold stress on their performance. The positive effect of curcumin on growth performance and FCR of broiler chickens was also reported by others (Ahmadi et al. 2010;Nouzarian et al. 2011). Curcumin by improving the uptake of digested nutrients (Hernandez et al. 2004) results in better feed efficiency and growth performance. Under normal temperature, the increase in dietary curcumin from 200 to 400 mg/kg caused a significant reduction in the growth of birds during the whole experimental period (61.35 vs 58.83 g/b/day, respectively). But, the growth performance of cold-stressed birds fed diet containing 400 mg/kg curcumin was better than that of those fed 200 mg/ kg curcumin (58.11 vs 57.76 g/b/day, respectively). It is proposed that the practical level of curcumin used for birds at normal temperature may not be adequate for cold-stressed birds. The negative effects of cold stress on birds' performance, as reported by others (Balog et al. 2003;Luger et al. 2001), may be due to the increase in metabolic rate to use more nutrients for the heat production to maintain body temperature (Ipek & Sahan, 2006). Therefore, cold stress causes an increase in oxygen requirements (up to 185%) (Gleeson 1986) and oxygen shortage may induce cellular hypoxia and increase the production of free radicals (Bottje & Wideman 1995) and subsequently increase tissue requirement of antioxidants in cold-stressed birds. The performance of cold-stressed birds fed diet containing 200 mg/kg of nanocurcumin was similar to that of those on diet with 400 mg/kg curcumin (59.47 vs 58.11 g/b/day, respectively), which may be due to more absorption or higher bioavailability of nanocurcumin. There is a water layer on the surface of intestinal epithelial cells and thus all types of substances should pass this barrier (Smithson et al. 1981). In fact, curcumin in the form of simple powder is lipophilic and thus insoluble in the water layer, whereas the The temperature of both houses were the same in the first two weeks; the temperature of one house was continued according to the recommended temperature, whereas the temperature in the second house was dropped to 15°C on day 14 and maintained between 13-15°C until day 42. micellization form is hydrophilic and can easily be solved in the water layer.
Due to the presence of polyunsaturated fatty acids, the membrane lipids are highly sensitive to oxidative damage (lipid peroxidation) (Halliwell & Gutteridge 1985) and measuring the oxidative damage in cell membrane lipids is one of the best ways to measure the effects of free radicals (Lykkesfeldt & Svendsen 2007) produced by oxidative reactions. The involvement of oxidative stress in ascites in broilers has been reported Bottje & Wideman 1995). Cold stressincreased plasma MDA was similar to the results reported by Fathi et al. (2011). In our study, the high level of MDA in coldstressed birds showed higher risk of ascites through oxidative damage. Similar to our results, Suvanated et al. (2003) reported that the use of turmeric in diet reduced TBA (thiobarbituric acid) indicator and improved antioxidant enzyme activity in broiler chickens compared with those fed control diet. Phenolic groups in the structure of curcumin have an important role in the prevention of lipid peroxidation. These groups can remove hydroxyl radical, superoxide ion and nitric oxides (Sreejayan & Rao 1996). Curcumin can reduce lipid peroxidation by the increase in antioxidants enzyme activity including glutathione peroxidase, superoxide dismutase or Catalase (Reddy & Lokesh 1996). But plasma MDA in birds was significantly increased when curcumin/nanocurcumin supplementation was increased from 200 to 400 mg/kg in diet. Donatus et al. (1990) reported that low concentration of curcumin can protect the liver against lipid peroxidation induced with paracetamol. But lipid peroxidation was increased when the concentration of used curcumin was increased to 100-fold because antioxidants may have the role of peroxidants depending on the dosage (Halliwell 2000). Therefore, the usage of high dose of curcumin/nanocurcumin results in induced lipid peroxidation and increased MDA.
Similarly, the effect of cold stress on increased AST/LDH activities in birds was reported by Fathi et al. (2011). Increased activities of AST/LDH is an indicator of advanced liver cells damage followed by increased production of free radicals resulting in the oxidative reactions chain in the liver and other organs (Arab et al. 2006). Increased production of free radicals during tissue hypoxia (Chen & Meyrick 2004) can negatively affect the activity of energy synthesis. In an anaerobic condition, LDH contributes to energy synthesis by anaerobic glycolysis. Hence, increased activity of LDH in cold-stressed birds is probably due to oxidative stress. On the contrary, dietary supplementation with turmeric increased AST and LDH activities in broiler chickens (Emadi & Kermanshahi 2007). Decreasing effects of curcumin on liver TBA indicated that curcumin can protect liver cells against free radicals attack through scavenging or neutralized free radicals (Akila et al. 1998). Therefore, decreased liver enzymes activities in this study may indicate improved liver function following the antioxidant effects of curcumin in the prevention of liver damage due to free radicals. In the present study, birds fed diet supplemented with nanocurcumin had higher liver enzymes activities (AST/LDH) than those fed curcumin-supplemented diet. It has been reported that curcumin in the form of nanoparticles (micellarcurcumin) has higher bioavailability, tissue distribution and biological half-life (Ma et al. 2007) than native curcumin. The high level of used nanocurcumin and its role as peroxidants (Halliwell 2000) result in induced liver cell injury and increased liver enzyme activities.
The effect of cold stress on increased plasma cholesterol in our study was in agreement with the results of Houshmand et al. (2012) who reported that stressors increased plasma cholesterol concentrations in broiler chickens. We observed that the supplementation of diet with curcumin significantly decreased Tchol, LDL and TG and increased HDL in the plasma of birds compared to that in those fed control diet. Different results on the effect of curcumin on Tchol/TG were reported by others, including a decrease in TG, Tchol and LDL in rat serum (Kim & Kim 2010), a decrease in liver TG without any effect on Tchol observed by the addition of 0.2% curcumin to diet (Manjunatha & srinivasan 2006), a decrease in TG with no effect on Tchol and LDL/HDL cholesterol (Nouzarian et al. 2011) or no change in Tchol, LDL and TG (Mehala & Moorthy 2008). The inconsistent results of turmeric on Tchol or TG may be attributed to the difference in the amount of curcumin in the used turmeric powder or the amount of curcumin may vary a lot between the various experiments. The conversion of cholesterol to bile acids in the liver is an important way to remove cholesterol from the body (Zhang et al. 2009). This conversion is a multistep process for which cholesterol-7-alpha-hydroxylase is the first stage in limiting the reaction rate. Because the animals fed diet supplemented with curcumin may have higher cholesterol-7-alpha-hydroxylase activity (Srinivasan & Sambaiah 1991), therefore decreasing the effects of curcumin on cholesterol may be through stimulating liver cholesterol-7hydroxylase activity. However, the reducing effect of curcumin on TG may be due to curcumin binding with TG or through the disruption in the lipid micellation, absorption and finally their disposal (Kim & Kim 2010). It is proposed that if arterial LDL oxidation causes atherosclerosis, then co-antioxidants may be anti-atherosclerotic (Witting et al. 1999). It is reported that turmeric extract reduced the susceptibility of LDL to oxidation in rabbits (Ramírez-Tortosa et al. 1999). Curcumin, which has antioxidant effects in addition to anti-hypercholestromic effects, can prevent atherosclerosis by protecting LDL from oxidation. In our study, the amount of Tchol, TG and LDL in the plasma of birds fed diet supplemented with nanocurcumin was significantly higher than that in those fed curcumin. Ramírez-Tortosa et al. (1999) reported that rabbits fed with 1.6 mg/kg body weight of turmeric extract had lower LDL than the control group. But rabbits fed with 3.2 mg/kg body weight of turmeric extract (two fold) had higher TG/LDL than those fed with 1.6 mg/kg or the control group.
In the present study, H/L ratio significantly increased in coldstressed birds. Under stress condition, the amount of corticosterone secretion increases from the adrenal glands, and is known to have a negative correlation between plasma corticosterone levels and lymphocyte proliferation (Hangalapura et al. 2004). But antioxidants prevent the proliferation of H, which are strong phagocytic cells, by a decrease in oxidative stress (Surai 2002). Similarly, the positive effect of curcumin in decreasing the number of H or H/L ratio was reported by Kijparkorn and Angkanaporn (2003). Curcumin increases the number of B and T lymphocytes and antibody production (Surh 1999;Gautam et al. 2007). Increased B and T cells in the intestine of rats fed with curcumin (1 g/kg) indicate that curcumin can improve immune function through lymphocytes proliferation (Churchill et al. 2000). The H/L ratio in birds fed diet supplemented with nanocurcumin was significantly higher than that in those fed curcumin. This may be related to the peroxidant activity of curcumin through phenoxyl radical production by the system of hydrogen peroxide peroxidase due to the oxidation of cellular glutathione or NADH and participation in the conversion of oxygen to ROS (Galati et al. 2002) which exacerbates the effect of stress on birds and increases H/L ratio.
Cold stress had a negative effect on jejunal histomorphological parameters (Figure 1). Stress condition increases the corticosterone secretion and thus dietary corticosterone supplementation (30 mg/kg diet) decreased the proliferation of jejunal epithelial cells and then decreased VH (Hu & Guo 2008). Our results showed that curcumin/nanocurcumin supplementation improved jejunal VH and VS. Similarly, the positive effect of curcumin on jejunal morphology was observed by Rajput et al. (2013) who reported that VH and VH/CD were improved in birds fed diet supplemented with curcumin (150 or 200 mg/kg) at 21 day of age compared to that in those fed 100 mg/kg or control diet. The epithelial cells of the intestine originate in the crypt and migrate to the villus tip (Potten 1998). The effect of curcumin on increased VH may be due to increased intestinal epithelial cell turnover, which is associated with activated cell mitosis (Samanya & Yamauchi 2002), and then longer villus, which can provide more VS and enhance nutrient absorption (Awad et al. 2008), may improve broiler chickens performance. In our study, there was no significant difference between the effect of curcumin/nanocurcumin on jejunal morphology parameters.
The number of E. coli in the caecum of cold-stressed birds was higher than that in those under normal condition. However, cold stress significantly decreased the number of lactobacilli. In stress condition, profitable microflora, especially lactobacilli, tends to decrease and harmful species have a tendency to increase. This phenomenon may reduce production parameters such as growth and feed efficiency (Fuller 2001). Dietary curcumin significantly decreased E. coli population in the caeca. Curcumin can increase bacterial cell membrane permeability. Thus, damage to the bacterial membrane is a key mechanism of curcumin in killing E. coli (Tyagi et al. 2015). Increased lactobacilli count in the caeca of birds fed diet supplemented with curcumin/nanocurcumin was observed in the present study. Viveros et al. (2011) reported that the effect of polyphenol compounds on the growth of some bacteria may be due to the ability of these bacteria to use phenolic compounds as food substrate. Lactobacilli can provide their energy requirements through the metabolism of phenolic compounds (Garcia-Ruiz et al. 2008). The effects of nanocurcumin on decreased E. coli population and increased lactobacilli population were higher than that of curcumin in our study. The higher activity of nanocurcumin compared to curcumin could be related to its particle size. The smaller particle size of nanocurcumin (2-40 nm) compared to curcumin (500-800 nm) is probably responsible for the better permeability and higher absorption into bacteria (Bhawana et al. 2011).

Conclusion
There was a beneficial effect of dietary supplementation of 200 mg/kg curcumin/nanocurcumin on chickens' growth performance at normal temperature. Unlike curcumin, coldstressed birds fed 200 mg/kg nanocurcumin had similar weight gain compared to those fed control diet and reared under normal temperature (59.5 vs 59.7 g/b/day). Also, it is revealed that dietary curcumin supplementation reduced plasma MDA, Tchol and TG and improved liver enzyme activities, immune system, jejunal morphology and microbial population. The effect of nanocurcumin on different measured parameters was similar or lower than that of curcumin with the exception of microbial population, which might be due to the high dose of nanocurcumin used in our study. Future studies with less amount of nanocurcumin (<200 mg/kg) may be helpful to point out the suitable level of nanocurcumin for the poultry industry.