Effect of Dietary Curcumin on the Antioxidant Status of Laying Hens under High-Temperature Conditions

ABSTRACT

Heat stress induces oxidative stress, and reduces body antioxidant metabolite levels, which can affect poultry production performance. Dietary antioxidants protect birds against the adverse effects of heat stress. The effects of increasing concentrations of dietary curcumin on the antioxidant parameters of layers maintained under high-temperature conditions for nine weeks were evaluated. Roman laying hens (n = 336, 22 weeks old, 1420 g BW) were divided into three treatment groups. The first group served as a thermoneutral control (kept at 25 ± 1 °C). The second group was exposed to high temperatures (32 ± 1 °C, 6 h/d), given a basal diet. The third group was further divided into five treatment groups (100, 150, 200, 250, 300 mg/kg Curcumin) fed a basal diet (treatments H1, H2, H3, H4, H5) under high temperatures conditions (32 ± 1 °C, 6 hours/day). As a result of this study, total superoxide dismutase activity was significantly higher in H2 and H3 groups, and total antioxidant capacity was higher in H2, H3, and H5 groups. Catalase and glutathione peroxidase activity was significantly higher in the H3 group. Malondialdehyde concentration was lowered in curcumin supplemented hens compared with control groups hens. Laying hens in all curcumin treatment groups had slightly higher activities of CAT, SOD, GSH-Px, and T-AOC in the liver, heart, and lungs, compared with heat stressed control group. It was concluded that dietary curcumin given to laying hens under heat stress may enhance their antioxidant status, and alleviate the detrimental effects of stressful environmental conditions.

Keywords:
Antioxidant; curcumin; heat stress; laying hen; oxidative damage

INTRODUCTION

In hot climatic regions of the world, heat stress (HS) causes oxidative stress in poultry (Lin et al., 2006; Habibi et al., 2014). Oxidative stress causes tissue damage and impair disease resistance, resulting in performance losses (Dhanalakshmi et al., 2007; Rahmani et al., 2017; Nawab et al., 2018). In southern China, in particular, high environmental temperatures disturbs poultry thermoregulation due to long duration of summer. Thermogenic mechanisms depend on fast metabolism in birds (Guo et al., 2007). However, fast metabolism rates increase the oxygen demand in the body tissues (Rahmani et al., 2017). Stressful conditions create an imbalance between oxygen demand and supply, thereby resulting in hypoxemia (Hassanzadeh et al., 2014). Hypoxia increases the production of free radicals (Reis et al., 2013), which in turn increases the activities of circulating enzymes, disturbing normal body functions (Arab et al., 2006; Rahmani et al., 2017).

Antioxidants protect cells from the effects of lipid peroxidation. Lipid peroxidation is an indicator of cellular injury due to generation of free radicals (Dinkova-Kostova & Talalay 2008; Wu et al., 2016). Several synthetic antioxidants have been banned due to their liver-carcinogenicity properties (Rahmani et al., 2017). On the other hand, several studies have reported that plant substances included in animal feeds provide the beneficial effects, including antioxidant action, activation of immune responses, as well as stimulation of appetite and improvement of endogenous digestive enzyme secretion (Ledoux, 2009; Toghyani et al., 2011; Nawab et al., 2018). Recently, curcumin, a yellow pigment of turmeric, has been considered a potential natural antioxidant feed additives (Wang et al., 2015; Ramos et al., 2017). Turmeric (Curcuma longa L) belongs to ginger family and is found in the southern and southeastern Asia (Nouzarian et al., 2011).

Curcumin is the main compound present in of turmericplant (Nouzarian et al., 2011; Wang et al., 2015; Arslan et al., 2017), and has been shown to have a wide range of therapeutic and pharmacological properties, including antioxidant, anti-inflammatory, free-radical scavenging, lipid peroxidation inhibition, antimicrobial, antiviral, antiprotozoal, and antitumor activities. In addition, turmeric may act boost the immune response (Cleary & McFeeters, 2006; Singh et al., 2010; Zhang et al., 2014; Wang et al., 2015; Pulido-Moran et al., 2016; Amalraj et al., 2017). Those curcumin biological properties make it a potential antioxidant feed additive for poultry. Therefore, the aim of our research was to evaluate the effects of increasing levels of dietary curcumin on antioxidant status of laying hens maintained under high environmental temperature conditions to determine if the harmful effects of heat stress could be alleviated.

MATERIALS AND METHODS

Birds, housing, experimental design, and diets

A total of 336 day-old Roman layers were purchased from Guangzhou poultry industry in Guangzhou, China. Birds were transported to the Department of Animal Sciences, Agricultural College, Guangdong Ocean University, where they were housed in battery cages (2 hens/cage), equipped with hopper feeder and nipple or cup drinker. Hens were provided with water and feed ad libitum throughout the experimental period.

Hens were evaluated from 22 (1420 g body weight) to 31 weeks of age (1940 g body weight). Hens were kept in an environmentally-controlled room (with controlled temperature, humidity, and light conditions until 22 weeks of age. In week 22, hens were divided into the following treatment groups: thermoneutral control group (TC; n=84), heat stress control group (HC; n=84) and heat stress treatment groups (HT; n= 84) named as H1, H2, H3, H4, H5. The thermoneutral environment was characterized by comfort temperatures of 25 ± 1 °C and 45-55% relative air humidity (RH). Heat stress was characterized by submitting birds to 32 ± 1 °C for 6 hours/day, between 10:00 and 16:00 h and 55-65% RH for nine weeks. Hens in the TC group were fed a basal diet formulated to supply layer nutritional requirements according to the NRC, (1994) (Table 2). The hens in the heat stress control group were fed the same basal diet as the TC hens, with no curcumin addition, and heat stress treatment group were given basal diet supplemented with curcumin at 100, 150, 200, 250, 300 mg/kg of feed, corresponding to treatments H1, H2, H3, H4, H5, respectively. The experimental treatments are described in Table 1.

Table 1
Diets fed to laying hens in this study.

Treatments	Abbreviations	Diet
Thermo-neutral control	TC	Basal diet
Heat control	HC	Basal diet
Treatment 1	H1	Basal diet + 100mg/kg curcumin
Treatment 2	H2	Basal diet + 150mg/kg curcumin
Treatment 3	H3	Basal diet + 200mg/kg curcumin
Treatment 4	H4	Basal diet + 250mg/kg curcumin
Treatment 5	H5	Basal diet + 300mg/kg curcumin

Table 2
Ingredients and chemical composition of the experimental basal diet.

Ingredients	Percent (%)	Nutrients (Analyzed composition, %)1	Content
Corn CP 8%	62.8	ME /(MJ/kg) 1	11.42
Soybean meal CP 44%	20.0	CP, %	18.17
Wheat bran 12%	2.0	Ca, %	3.7
Fish meal CP 62%	4.5	TP, %	0.58
Limestone, %	9.0	Met, %	0.41
CaHPO 4%	1.0	Cys,%	0.29
NaCl, %	0.2	Lys, %	0.94
Premix1	0.5

• Notes: ¹The premix provided, per kg of diet: Vitamin A 9000 IU; Vitamin D 2500 IU; Vitamin E 20 IU; Vitamin B 1212µg; Vitamin K 2.4 mg; Mn100 mg; Zn 60 mg; Fe 25 mg, Cu 5 mg; Co 0.1 mg (Mn, Zn, Fe, Cu, Co were provided in the form of sulfates); Se (N2SeO3.5H2O) 0.2 mg; I(KI) 0.5mg.
• ¹
  Calculated values were according to NRC (1994) values for feedstuffs.

Dietary curcumin was obtained from Agricultural Vegetable Limited Company in Xi’an, China. Curcumin was composed of 77% curcumin, 18% dimethoxy curcumin, and 5% bisdemethoxy curcumin (Rahmani et al., 2017). The purity of the curcumin used in this study was 95%. Curcumin was first added to a small amount of basal diet and then thoroughly mixed with 100 kg feed at the required amounts to obtain the HS1, HS2, HS3, HS4, and HS5 diets.

Blood sampling and determination of antioxidant enzyme activities

Blood samples were taken from the wing vein of three randomly-selected hens fasted overnight per replicate during the 3^rd, 6^th and 9^th week of the experiment. Each sample was collected in two tubes (one with and one without EDTA as an anticoagulant). Blood samples were kept at room temperature for 45 min, and the serum was obtained by centrifugation at 700 g for 10 min. Serum was stored in 2 mL plastic vials at -20 °C for further analysis.

Liver, lung, and heart tissue samples were also collected for measurement of antioxidant enzymatic activities, and stored at -20 °C. The tissue samples were prepared in PBS (phosphate buffered saline) buffer, and centrifuged at 2,500 g for 10 min at 4 ºC. The assays were conducted according to the procedures described by Wang et al. (2015). Serum catalase (CAT) activity was assessed by the method described by Sippy et al. (2003) (ELISA kit: QuantiChrom, BioAssay Systems, USA, Catalog No. ECAT-100). Superoxide dismutase (SOD) activity was measured using the xanthine and xanthine oxidase method (ELISA kit: Cayman Chemical Company, USA, Catalog No. 706002), which measures the inhibition of the nitroblue tetrazolium reduction reaction in extracts of the collected tissue samples(Sun et al. 1988). Serum glutathione peroxidase (Gpx) was measured using H₂O₂and a specific dye containing an electron donor that results in a pink color during the peroxide reaction (ELISA Kit: QuantiChrom, Bioassay Systems, USA, Catalog No. DPOD-100), following Kokkinakis & Brooks (1979). Serum total antioxidant capacity (TAC) was measured by using a Randox total antioxidant status kit (Randox Laboratories Ltd, Crumlin, UK). Serum malondialdehyde (MDA) levels, as an indicator of lipid peroxidation, were determined by using thiobarbituric acid reactive substances (TBARS) produced during oxidative stress (Ohkawa et al. 1979) (ELISA Kit: QuantiChromTM, Bioassay Systems, USA, Catalog No. DTBA-100), according to Ohkawa et al. (1979). The assays were conducted according to the manufacturers’ protocols.

Statistical analysis

Statistical analysis was carried out using (SPSS Statistical Software, 1968). Data were submitted to one-way analysis of variance, and means compared by Duncan’ significant difference test (Steel et al., 1997). All data are expressed as means ± standard error (SEM). Results were considered statistically significant at p<0.05.

RESULTS AND DISCUSSION

Several stress factors cause the generation of reactive oxygen species (ROS), such as superoxide (O₂) and hydrogen peroxide (H₂O₂), which lead to oxidative stress (Zeng et al., 2014). Oxidative stress can be described as an imbalance between pro-oxidant and antioxidant metabolites (Daneshyar, 2012; Ismail et al., 2013). Elevated levels of ROS can overwhelm cellular homeostasis by initiating lipid peroxidation, oxidation of proteins, and inhibition of enzymes, that ultimately lead to cell death (figure 1)(Maheshwari & Dubey, 2009; Srivastava & Dubey, 2011; Zeng et al., 2014). Antioxidant metabolites (SOD, CAT, T-AOC, and GSH-Px) play the role of defense mediators in animal bodies (Daneshyar, 2012). Decreased or increased concentrations of antioxidant metabolites and free radicles (ROS) have detrimental effects on body tissues, which in turn, result in the manifestation of diseases (Wang et al., 2015). MDA is one of main products of lipid peroxidation, and can be monitored by determining MDA concentrations in serum and tissue samples (Wang et al., 2015).

Figure 1
Effect of heat stress on poultry health.

Curcumin was shown to present excellent antioxidant and anti-inflammatory activities (Yarru et al., 2009; Nouzarian et al., 2011; Rahmani et al., 2017). Curcumin is a main antioxidant element of turmeric plants (Cousins et al., 2007; Wang et al., 2015). It has the specific ability to scavenge free radicals (including superoxide anions and hydroxyl radicals) and hinder lipid peroxidation (Yarru et al., 2009; Wang et al., 2015).

Studies have reported that curcumin given to rats had inhibited lipid peroxidation in liver microsomes and erythrocyte membranes (Chattopadhyay et al., 2004; Wang et al., 2015). In our experiment, positive effects of curcumin on the antioxidant and oxidant status of serum and tissues samples of laying hens submitted to heat stress for nine weeks were detected (Figure 2). Serum SOD activity was significantly (p<0.05) higher in hens maintained in TC compared with HS hens fed no curcumin in all evaluated weeks. SOD activity was significantly (p<0.05) increased in heat-stressed groups fed curcumin at 150 and 200 mg/kg (HT150 and HT200) in the 3^rd and 9^th week of the experiment, respectively, compared with the TC and HC group, which were fed only the basal diet. In the 6^th week of the experiment, heat-stressed hens supplemented with all curcumin levels (HT100, HT150, HT200, HT250, HT300 mg/kg) had increased serum SOD activity, but results were not statistically significant (p>0.05). These results were in accordance with Daneshyar (2012).

Figure 2
Effect of dietary curcumin on poultry performance.

Table 3 presents antioxidant metabolite activities determined in the serum of the experimental hens. Serum T-AOC activity was significantly (p<0.05) higher in curcumin-fed hens in a dose-dependent manner compared with the heat stressed control group (not supplemented with curcumin) during weeks 3, 6, and 9 of the experiment, as previously discussed by Daneshyar (2012). Serum T-AOC activity was significantly (p<0.05) enhanced in groups HT200, HT150, and HT250 during the 3^rd, 6^th, and 9^th week of the experiment, respectively, compared with the HC group, fed only the basal diet. Serum CAT activity was significantly (p<0.05) increased in the serum samples of hens in heat-stressed curcumin supplemented groups HT150 and HT200 during week 3 and HT200 during week 9 of the experiment when compared with the heat stress (HC) control group, which is in accordance with Wang et al. (2015). On the other hand, in 6 week of the experiment, heat stress curcumin supplemented groups (HT100, HT150, HT200, HT250 and HT300 mg/kg) showed significantly (p<0.05) higher serum CAT activity compared with the HC group, given only the basal diet.

Table 3
Serum antioxidant metabolites of control hens (TC; no heat stress, no curcumin), heat stressed hens not supplemented with dietary curcumin (HC) and heat stressed hens supplemented with different concentrations of dietary curcumin (H1, H2, H3, H4, H5, for concentrations see Table 1) for nine weeks. Data are shown as mean ± SEM.

Parameters1	Time (Week)	TC	HC	H1	H2	H3	H4	H5
SOD (U/mL)	3	43.8±2.83a	40.60±3.32b	42.36± 3.00ab	43.44± 2.81a	41.12± 2.56ab	40.70± 2.59b	43.03± 2.55ab
	6	43.2± 3.25a	35.31± 5.76b	39.01± 4.00ab	41.84± 4.13ab	41.78± 2.16ab	38.06± 4.63ab	38.40± 4.26ab
	9	60.8± 4.95a	51.11± 3.56b	61.31± 5.38a	63.91± 3.30a	63.32± 5.07a	58.31± 5.47ab	57.47± 4.05ab
CAT (U/mL)	3	7.03± 0.23ab	5.30± 0.50b	7.76± 0.29ab	10.01± 0.56a	8.79± 0.40a	7.67± 0.67ab	5.97± 0.34ab
	6	7.57±0.42b	4.81± 0.66a	6.93± 0.31b	6.52± 0.27b	6.76± 0.37b	6.24± 0.39b	6.21± 0.37b
	9	10.1± 0.82ab	6.30± 0.36c	8.48± 0.71b	9.96± 0.78ab	12.43± 3.42a	9.87± 0.73ab	9.37± 0.95b
T-AOC (U/mL)	3	10.5± 4.17a	5.59± 2.50b	6.16± 4.43b	9.57± 2.49ab	5.94± 2.74b	6.25± 1.04b	9.06± 1.78ab
	6	4.89±1.53	3.00± 1.23	4.77± 2.96	4.93± 1.66	4.32± 1.83	4.23± 2.31	4.23± 1.63
	9	4.54± 1.87	2.07± 1.43	4.36± 1.68	3.42± 0.82	3.55± 2.22	5.15± 2.87	4.08± 1.29
GSH-Px (U/mol)	3	262.2±34.81ab	233.29±12.56c	239.58±19.11c	240.92±12.14c	287.71±33.26a	275.52±16.15a	248.98±18.45bc
	6	324.2±12.39a	297.7±88.11a	313.7±89.66a	288.5±76.83a	551.0±148.25b	328.5±48.96a	288.5±59.99a
	9	321.22±12.39ab	277.66±48.15a	283.25±87.23ab	278.20±46.43a	451.50±98.33b	329.50±78.66ab	278.50±59.99a
MDA (nmol/mL)	3	15.6± 5.53b	18.58±4.24a	17.38± 4.15ab	17.75± 5.17ab	17.42± 7.66ab	16.84± 7.73ab	16.38± 4.52ab
	6	13.00± 1.33b	15.77±0.19a	13.56± 1.83ab	13.99± 0.58ab	13.67± 1.15ab	13.11± 1.35ab	13.67± 1.15ab
	9	14.0± 1.53b	16.22±1.07a	15.00±0.88ab	15.67± 0.58ab	15.78± 1.02ab	14.83± 0.88ab	15.67± 1.15ab

• Note: Numbers with different lowercase letters are significantly different from each other (p<0.05). Numbers not followed by different lowercase letters are not significantly different from each other (p>0.05).
• ¹
  SOD = superoxide dismutase; CAT = catalase; T-AOC = total antioxidant capacity; GSH-Px = glutathione peroxidase; MDA = malondialdehyde.

Furthermore, GSH-Px activity was also significantly (p<0.05) higher in the serum samples of curcumin-fed HT200 group in the 3^rd and 6^th weeks of the experiment, whereas in the 9^th week, curcumin supplementation at 200 and 250 mg/kg (groups HT200 and HT250) increased GSH-Px activity compared with the non-supplemented HC and TC groups. In addition, despite not statistically different (p>0.05), serum MDA levels were lower in all heat-stress groups fed the diets supplemented with curcumin in weeks 3, 6, and 9 of the experiment compared with the heat-stressed group fed the basal diet. Similar findings were reported by Cousins et al. (2007) and Daneshyar (2012).

Wang et al. (2015) indicated that dietary curcumin counteracts the process of lipid peroxidation and reduces the production of reactive free radicals which, in response, increase the concentrations of antioxidant metabolites in the poultry body. In the present experiment, hens in all curcumin-supplemented groups (H1, H2, H3, H4, H5) had slightly higher activities of CAT, SOD, GSH-Px, and T-AOC in the liver, heart, and lung tissues compared with the-heat stressed control group (Wang et al., 2015).

Table 4 shows the results of antioxidant metabolites determined in the liver of the experimental birds. SOD activity was significantly (p<0.05) increased in the 3^rd week (treatmentsH2 and H5), 6^th week (treatmentsH2 and H3), and 9^th week (treatment H4) of the experiment compared with HC group fed only the basal diet. CAT activity was significantly (p<0.05) increased in liver in the 3^rd week (treatment H4), 6^th week (treatment H3 and H4), and 9^th week (treatment H4) of the experiment compared with the HC group fed only the basal diet. T-AOC activity was also significantly (p<0.05) increased in the 9^th week (treatment H3) of the experiment compared with the HC group fed only the basal diet; however, in the 3^rd and 6^th weeks of the experiment, the curcumin-supplemented diet fed to heat-stressed hens had no significant (p>0.05) effect on T-AOC activity. GSH-Px activity was also significantly (p<0.05) increased in the liver of heat-stressed hens fed curcumin-supplemented diets in the 3^rd week (treatment H3), 6^th week (treatment H4), and 9^th week (treatment H4) of the experiment compared with the HC group fed only the basal diet.

Table 4
Assay of antioxidant metabolites in liver tissue of control hens (TC; no heat stress, no curcumin), heat stressed hens not supplemented with dietary curcumin (HC) and heat stressed hens supplemented with different concentrations of dietary curcumin (H1, H2, H3, H4, H5, for concentrations see Table 1) for nine weeks.

Parameters1	Time (Week)	TC	HC	H1	H2	H3	H4	H5
SOD (U/mL)	3	143.88±22.83	140.60±32.32	142.36±32.00	143.44±22.81	141.12±22.56	140.70±22.59	143.03±23.55
	6	143.20±33.25	135.31±53.76	139.01±43.00	141.84±43.13	141.78±23.16	138.06±42.63	138.40±42.26
	9	160.81±42.95	151.11±33.56	151.31±25.38	153.91±33.30	153.32±53.07	158.31±53.47	157.47±42.05
CAT (U/mL)	3	19.32±1.51a	10.64±5.74b	13.66±6.53ab	14.75±1.32ab	16.74±0.99ab	19.02±0.52a	11.49±0.87b
	6	26.66±0.17a	15.41±4.20bc	20.18±3.76b	18.29±3.38b	18.88±5.94b	20.64±2.40b	14.21±0.98c
	9	21.61±4.92	18.20±5.36	21.48±3.26	18.84±4.85	18.26±6.15	22.17±5.77	20.30±4.45
T-AOC (U/mL)	3	8.54±1.17	7.01±1.50	7.16±1.43	7.57±1.9	6.65±1.74	7.25±2.04	7.56±1.23
	6	7.29±1.53a	4.25±1.33b	5.77±2.56ab	5.55±1.36ab	6.32±1.83ab	5.53±1.31ab	4.26±1.63b
	9	6.67±1.7	5.58±1.43	5.70±0.68	6.02±0.2	7.55±3.2	5.85±2.87	5.08±1.9
GSH-Px (U/mol)	3	162.29±34.81	133.29±12.56	139.58±19.11	140.92±12.14	187.71±43.26	175.52±16.15	158.98±18.45
	6	194.62±12.39	197.75±88.11	153.75±49.66	188.50±76.83	151.00±48.25	228.50±48.96	188.50±59.99
	9	191.22±12.39	177.66±48.15a	183.25±67.23	178.20±46.43	151.50±58.33	229.50±68.66a	178.50±59.99
MDA (nmol/mL)	3	3.67±0.53b	4.78±0.24a	3.98±0.05ab	3.75±5.17ab	3.42±7.66ab	4.04±1.73ab	4.38±0.42ab
	6	4.00±1.33b	6.07±1.19a	4.90±1.33ab	5.49±0.56ab	4.67±2.11ab	7.11±0.75a	5.67±1.45ab
	9	6.55±4.53b	8.22±2.07a	8.01±2.88a	7.37±1.78ab	7.78±1.08ab	6.83±0.88ab	9.67±1.45a

• Note: Numbers with different lowercase letters are significantly different from each other (p<0.05). Numbers not followed by different lowercase letters are not significantly different from each other (p>0.05).
• ¹
  SOD = superoxide dismutase; CAT = catalase; T-AOC = total antioxidant capacity; GSH-Px = glutathione peroxidase; MDA = malondialdehyde.

Antioxidant metabolite activities assayed in the heart are shown in Table 5. SOD activity was significantly (p<0.05) increased in heat-stressed hens fed curcumin-supplemented diets in the 6^th week (treatments H2 and H3) and 9^th week (treatmentsH2 and H3) compared with the HC group fed only the basal diet, but no statistical differences (p>0.05) were detected in the 3^rd week. CAT activity was significantly (p<0.05) increased in heat-stressed hens fed curcumin-supplemented diets in the 3^rd week (treatment H3), 6^th week (treatments H1 and H5), and 9^th week (treatment H5) of the experiment compared with the HC group fed only the basal diet. Heart T-AOC activity was also significantly (p<0.05) increased in the 3^rd week (treatments H3 and H4) and 6^th week (treatments H1 and H2) of the experiment compared with the HC group fed only the basal diet, but in the 9^th week, curcumin supplementation did not significantly (p>0.05) affect T-AOC activity. Moreover, heart GSH-Px activity significantly (p<0.05) increased in the 3^rd week (treatments H3 and H4), 6^th week (treatmentsH3 and H5), and 9^th week (treatment H3) of the experiment compared with the HC group given only basal diet.

Table 5
Assay of antioxidant metabolites in heart tissues of control hens (TC; no heat stress, no curcumin), heat stressed hens not supplemented with dietary curcumin (HC) and heat stressed hens supplemented with different concentrations of dietary curcumin (H1, H2, H3, H4, H5, for concentrations see Table 1) for nine weeks.

Parameters1	Times (Week)	TC	HC	H1	H2	H3	H4	H5
SOD (U/mL)	3	243.88±2.83	240.60±3.32	242.36±3.00	243.44±2.81	241.12±2.56	240.70±2.59	243.03±2.55
	6	243.20±3.25	235.31±5.76b	239.01±4.00	241.84±4.13	241.78±2.16	238.06±4.63	238.40±4.26
	9	260.81±4.95	251.11±3.56	261.31±5.38	263.91±3.30	263.32±5.07	258.31±5.47	257.47±4.05
CAT (U/mL)	3	13.03±0.23a	4.00±1.04bc	2.76±0.29c	6.01±0.56bc	13.79±0.40a	5.67±0.67bc	7.97±0.34b
	6	11.57±0.2	9.81±0.66	12.93±0.1	10.52±0.7	10.76±0.37	11.24±0.39	12.21±0.7
	9	9.99±0.82ab	6.30±0.36b	8.88±0.71ab	9.96±0.78ab	10.43±3.42ab	8.87±0.73ab	12.37±0.95a
T-AOC (U/mL)	3	4.54±4.1b	3.59±2.5b	4.16±4.4b	5.57±2.4b	8.94±2.4a	9.25±1.4a	8.06±1.7a
	6	12.89±1.53a	5.00±1.23b	12.77±2.96a	12.93±1.66a	12.32±1.83a	9.23±2.31ab	9.23±1.63ab
	9	3.54±1.87	3.07±1.43	3.36±1.68	3.42±0.82	3.55±2.22	3.15±2.87	3.08±1.29
GSH-Px (U/mol)	3	262.29±34.81ab	233.29±12.56c	239.58±19.11c	240.92±12.14c	287.71±33.26a	275.52±16.15a	248.98±18.45bc
	6	324.62±12.39a	297.75±88.11a	313.75±89.66a	288.50±76.83a	551.00±148.25b	328.50±48.96a	288.50±59.99a
	9	321.22±12.39ab	277.66±48.15a	283.25±87.23ab	278.20±46.43a	451.50±98.33b	329.50±78.66ab	278.50±59.99a
MDA (nmol/mL)	3	5.67±5.53b	10.58±4.24a	7.38±4.15ab	8.75±5.17ab	8.42±7.66ab	6.84±7.73ab	9.38±4.52a
	6	5.00±1.3	5.77±0.19	5.56±1.3	5.99±0.8	5.67±1.5	5.11±1.5	5.67±1.5
	9	9.08±1.53	10.22±1.07	8.00±0.88	8.67±0.58	10.78±1.02	14.83±0.88	10.67±1.15

• Note: Numbers with different lowercase letters are significantly different from each other (p<0.05). Numbers not followed by different lowercase letters are not significantly different from each other (p>0.05).
• ¹
  SOD = superoxide dismutase; CAT = catalase; T-AOC = total antioxidant capacity; GSH-Px = glutathione peroxidase; MDA = malondialdehyde.

Table 6 presents the activities of antioxidant metabolites determined in the lungs of the experimental layers. Lung SOD activity was significantly (p<0.05) increased in the 3^rd week (treatment H2) and 9^th week (treatments H2 and H3) of the experiment in heat-stressed hens fed curcumin-supplemented diets compared with the HC group fed only the basal diet, but no significant (p>0.05) differences were determined in the 6th week. CAT activity was also significantly (p<0.05) increased in the lungs of hens submitted to heat stress and fed curcumin in the 3^rd week (treatmentsH2, H4 and H5), 6^th week (treatments H2, H4 and H5), and 9^th week (treatments H2, H3 and H4) of the experiment compared with the HC group fed only the basal diet. LungT-AOC activity of heat-stressed hens fed diets supplemented with curcumin was significantly (p<0.05) increased in the 3^rd week (treatments H2 and H3) and 9^th week (treatmentsH3, H4, and H5) of the experiment compared with the HC group fed only the basal diet, but was not significantly (p>0.05) different in the 6^th week. Furthermore, lung GSH-Px activity was also significantly (p<0.05) increased in heat-stressed hens fed curcumin in the 3^rd week (treatmentsH3 and H5), 6^th week (treatment H1), and 9^th week (treatment H3 and H4) of the experiment compared with the HC group given only the basal diet.

Table 6
Assay of antioxidant metabolites in lung tissues of control hens (TC; no heat stress, no curcumin), heat stressed hens not supplemented with dietary curcumin (HC) and heat stressed hens supplemented with different concentrations of dietary curcumin (H1, H2, H3, H4, H5, for concentrations see Table 1) for nine weeks.

Parameters1	Time (Week)	TC	HC	H1	H2	H3	H4	H5
SOD (U/mL)	3	86.24±2.32a	80.20±3.32b	84.36±3.22ab	86.44±2.22a	82.12±2.23ab	80.70±2.32b	85.03±2.55ab
	6	88.20±3.25a	70.31±5.76b	78.01±4.00ab	82.84±4.13ab	82.78±2.16ab	76.06±4.63ab	76.40±4.26ab
	9	100.81±4.95a	81.11±3.56b	91.31±5.38a	93.91±3.30a	93.32±5.07a	88.31±5.47ab	87.47±4.05ab
CAT (U/mL)	3	18.03±0.23a	8.01±1.4c	7.74±1.20c	16.01±0.56ab	12.79±0.40bc	16.67±0.67ab	16.97±0.34ab
	6	21.57±0.42a	4.85±2.6c	8.83±3.31c	22.52±0.27a	13.76±0.37b	19.24±0.39a	20.21±0.37a
	9	19.19±0.82a	8.30±0.36b	16.48±0.71ab	29.96±0.78a	22.43±3.42a	23.87±0.73a	16.37±0.95ab
T-AOC (U/mL)	3	2.54±1.17	1.59±0.50	1.16±0.43	2.57±2.49	2.94±0.74	1.25±1.04	1.06±0.78
	6	1.89±1.53	1.00±1.23	1.77±2.96	1.93±1.66	1.32±1.83	1.23±2.31	1.23±1.63
	9	6.54±1.87ab	4.07±1.43b	4.36±1.68b	5.42±0.82b	8.55±2.22a	9.15±2.87a	8.45±1.29a
GSH-Px (U/mol)	3	242.29±34.81	233.29±12.56	239.58±19.11	241.92±12.14	247.71±33.26	245.11±16.15	248.98±18.45
	6	304.62±12.39	297.75±88.11	313.75±89.66	208.50±76.83	211.00±148.25	218.50±48.96	208.50±59.99
	9	321.22±12.39	297.16±48.15	303.15±87.13	308.20±46.13	311.50±92.33	309.50±38.66	298.50±49.99
MDA (nmol/mL)	3	6.67±5.53	7.58±4.24	7.38±4.15	7.75±5.17	6.42±7.66	6.84±7.73	6.38±4.52
	6	5.00±1.33	4.77±0.19	4.56±1.83	4.99±0.58	4.67±1.15	4.11±1.35	4.67±1.15
	9	5.08±1.53	5.22±1.07	5.00±0.88	4.67±0.58	4.78±1.02	4.83±0.88	5.67±1.15

• Note: Numbers with different lowercase letters are significantly different from each other (p<0.05). Numbers not followed by different lowercase letters are not significantly different from each other (p>0.05).
• ¹
  SOD = superoxide dismutase; CAT = catalase; T-AOC = total antioxidant capacity; GSH-Px = glutathione peroxidase; MDA = malondialdehyde.

The concentration of MDA in the liver, heart, and lungs was decreased in all heat-stressed groups fed graded curcumin levels (H1, H2, H3, H4 and H5) compared with those submitted to heat stress or maintained at comfort temperature and fed only the basal diet (but not significantly), suggesting that dietary curcumin is capable of alleviating the deleterious effects of heat-stress, acting as an antioxidant (Wang et al., 2015). The significantly higher concentrations of MDA in the heat-stress control (HC) hens observed in our study was in accordance with reports from studies with heat-stressed broiler chickens (Zhang et al., 2009; Ledoux, 2009; Habibi et al., 2014; Wang et al.,2015) and laying hens (Akbarian et al., 2011). Interestingly, the significantly higher activities of CAT, SOD, T-AOC, and GSH-Px obtained in the heat-stressed hens fed diets supplemented with curcumin (200 and 250 mg/kg) suggest that curcumin may provide a protective mechanism against oxidative stress and lipid peroxidation (Wang et al., 2015). This indicates the potential of dietary curcumin to initiate the biosynthesis of antioxidant enzymes, as well as to reduce heat-stress induced oxidative damage (Yarru et al., 2009). The mechanism that describes how dietary curcumin can reduce the negative effects of heat stress may explain that stressful environmental conditions stimulate the secretion of corticosteroids, which can be counteracted by dietary curcumin supplementation.

Taken together, the results of our study suggested that laying hens fed dietary curcumin at 200 and 250 mg/kg had better heat tolerance compared with the control groups, which is reflected by higher activities of CAT, SOD, T-AOC, and GSH-Px, as well as lower MDA concentrations in the serum and tissue samples compared with the heat stress control group, which may help to protect the cells and tissues from lipid peroxidation. The results of present study also indicated that curcumin improves the antioxidant metabolites of birds, and can be a suitable feed additive as an alternative to synthetic antioxidants in the poultry diets which may enhance the bird’s immunity against stressful environmental conditions.

ACKNOWLEDGEMENTS

Special thanks to Revista Brasileira de Ciência Avícola for giving an opportunity to share knowledge on worldwide heat stress issues and strategies to overcome this problem. The author wishes to thank beloved parents (Rana Nawab Ahmad and Mrs. R. Sultana), grandparents (Saith Bachal Din), uncle (Rana Maqbool Ahmad), brother (Rana Kashif Nawab and Dr. Rana Yasir Nawab) and dearest all teachers for continued support and excellent mentorship. This work was supported by Science and Technology Planning Project of Guangdong Province, China (2010 B090400376).

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  According to rules and regulation of Guangdong Ocean University Animal Care and use Committee (Guangdong Province, China)

Publication Dates

• Publication in this collection
  14 Nov 2019
• Date of issue
  2019

History

• Received
  26 Oct 2018
• Accepted
  04 Feb 2019