Effects of genistein and hesperidin on biomarkers of ...

Effects of genistein and hesperidin on biomarkers of heat stress in broilers under persistent summer stress A. A. Kamboh,1 S. Q. Hang, M. Bakhetgul, and W-Y. Zhu1 Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China ratios were found to decrease (P < 0.01) in the treated groups. Moreover, biomarkers of heat stress including the level of creatine kinase, lactate dehydrogenase, and heat shock protein 70 mRNA of breast muscle was also changed (P < 0.01) positively by the dietary compounds with pronounced effects of combined treatments. These findings suggested that genistein and hesperidin could be a prime strategy to ameliorate summer stress effects in broilers; and a combination of both compounds may lead to mutual synergistic effects. It could be suggested that dietary use of both genistein and hesperidin as a feed supplement may offer a potential nutritional strategy in tropical and subtropical regions to overcome the deleterious effects of persistent summer stress in broiler production.

Key words: genistein, hesperidin, summer stress, biomarker, broiler 2013 Poultry Science 92:2411–2418 http://dx.doi.org/10.3382/ps.2012-02960

INTRODUCTION Summer stress is considered a serious issue of the poultry industry in tropical and subtropical regions of the world. Chickens subjected to high ambient temperatures not only show high mortality and low performance but also produce inferior quality meat (McCurdy et al., 1996; Woelfel et al., 2002). Researchers have declared that postmortem myopathy results from the combination of heat and acidosis produced in the skeletal muscles due to hypermetabolism; however, muscle hypermetabolism in animals is typically caused by antemortem stress factors, particularly by heat stress (Chiang et al., 2008). Antemortem heat stress induces several hormonal changes in warm-blooded animals, particularly in the thyroid hormones. Altered levels of thyroid hormones affect Ca2+ regulation of skeletal muscle and also influ-

©2013 Poultry Science Association Inc. Received December 5, 2012. Accepted May 11, 2013. 1 Corresponding authors: [email protected] and [email protected]

ence its expression in the myofibrillar proteins. During muscle contraction, presence of high Ca2+ contents leads to muscle hypermetabolism that could develop postmortem meat quality defects (myopathy) or cause rhabdomyolysis (disruption of skeletal muscle fibers; Chiang et al., 2008). The hyperthermia-associated myopathy is manifested by the altered activities of aspartate transaminase, lactate dehydrogenase (LDH), and increased activities of isoenzyme creatine kinase (CK; Poels and Gabreëls, 1993; Daroit and Brandelli, 2008). Heat stress in birds has also been associated with an increased number of heterophils and decreased number of lymphocytes and BW (Cīrule et al., 2012). Moreover, heat stress increases the expression of heat shock proteins (HSP) in skeletal muscle, which are universal cytoprotective proteins that enhance the tolerance to stress and increase the survival rate of stressed cells (Jacquier-Sarlin et al., 1994). Recent studies have shown that, supplementation of natural antioxidants is a promising strategy to minimize the deleterious effects of heat stress in farm animals, probably due to their quenching action on free radicals generated by heat stress. Studies in broilers (Patra et al., 2011) and quail (Imik et al., 2010) have shown that

2411

Downloaded from http://ps.oxfordjournals.org/ by guest on November 4, 2014

ABSTRACT This study investigated the supplemental effects of the flavonoids genistein and hesperidin for biomarkers of heat stress in broilers reared under persistent summer stress. A total of 360 one-day-old, mixed-sex broiler chickens were divided into 6 treatment groups: control or supplemented with 5 mg of genistein·kg of feed−1, 20 mg of hesperidin·kg of feed−1, or a mixture of genistein and hesperidin (1:4) at a dosage of 5 mg·kg−1, 10 mg·kg−1, and 20 mg·kg−1 of feed. Broilers were slaughtered at 42 d and samples were analyzed for hematological profile, creatine kinase, lactate dehydrogenase, and heat shock protein 70 mRNA levels. Results showed that dietary genistein and hesperidin improved (P < 0.05) the weekly performance of broilers particularly during the finisher period. The circulating heterophils and heterophil-to-lymphocyte

2412

Kamboh et al.

MATERIALS AND METHODS Birds and Diet A total of 360 Arbor Acre broiler chicks (1 d old) of mixed-sex were purchased from a commercial hatchery (Hewei, Anhui, PR China) and randomly divided into 6 treatment groups (6 replicates of 10 broilers per cage). The birds were fed corn-soybean basal diets that were prepared each week to keep fresh feed available to the broilers. Diet formulation and composition were adjusted according to the standard (NRC, 1994) and contained ME of 3.12 and 3.19 Mcal∙kg−1 in starter (1–21) and finisher (22–42) periods, respectively (Table 1). Broilers in the control group were fed with no additive, whereas other groups were fed 5 mg∙kg−1 of genistein (G5); or 20 mg∙kg−1 of hesperidin (H20); or a mixture of genistein and hesperidin (1:4) in doses of 5 mg∙kg−1 (GH5); 10 mg∙kg−1 (GH10), and 20 mg∙kg−1 (GH20), respectively. Purified flavonoids genistein and hesperidin were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO) with 98% purity. Their purity was further confirmed before their supplementation to broiler diets.

Table 1. Ingredients and composition of experimental diets for starter (1–21 d) and finisher (22–42 d) periods Item Ingredient (g∙kg−1) Corn Soybean meal Corn gluten meal Soybean oil Dicalcium phosphate Limestone Sodium chloride Lys Met Vitamin-mineral premix1 Chemical composition ME (Mcal/kg) CP (%) Ca (%) Lys (%) Met (%) Met + Cys (%) Total P (%)

Starter period

Finisher period

607 300 25 23 17 12 3 1.6 1.4 10

660 240 30 25.5 17 12 3 1.5 1.0 10

3.12 21.1 0.9 1.1 0.5 0.9 0.4

3.19 19.0 0.9 0.95 0.4 0.75 0.4

1Supplied per kilogram of diet: transretinyl acetate, 25 mg; cholecalciferol, 6 mg; menadione, 1.2 mg; thiamine, 2.3 mg; riboflavin, 8 mg; nicotinamide, 42 mg; choline chloride, 400 mg; calcium pantothenate, 10 mg; pyridoxine HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; cobalamin, 0.012 mg; Fe (from ferrous sulfate), 82 mg; Cu (from copper sulfate), 7.5 mg; Mn (from manganese sulfate), 110 mg; Zn (from zinc oxide), 64 mg; I (from calcium iodate), 1.1 mg; Se (from sodium selenite), 0.28 mg.

Husbandry The experiment was conducted during June to July at Animal Experimental Centre of Nanjing Agricultural University. All experimental procedures were approved by the Animal Ethics and Use Committee of Nanjing Agricultural University that adopted the Animal Care and Use guidelines. The broilers were provided 24 h of light from 0 to 3 d and 16L:8D from 4 to 42 d of age. The maximum and minimum room temperature for day and night ranged from 34.0 to 37.3°C and 24.4 to 27.8°C, respectively, whereas the average RH was 65 to 75% during the whole experimental period. All broilers had free access to feed and water during whole trial period of 42 d. As a routine practice, all broilers were vaccinated against Newcastle disease and infectious bursal disease. Feed consumption, BW, and mortalities were recorded for each pen on daily basis and used to calculate the weekly performance.

Sample Collection and Handling At 42 d, after a 12 h feed withdrawn, 2 broilers from each replicate (12 per group) were randomly selected and slaughtered through cutting of jugular veins and carotid arteries. Two-millimeter blood samples were collected in tubes containing anticoagulant heparin and kept on ice and immediately used for hematological analysis. Skinless breast meat (pectoralis major) samples were also collected and quickly snap-frozen in liquid nitrogen that was further used to analyze the biomarkers of heat stress including CK, LDH, and HSP70 level.


supplementation of vitamin C and E could counteract the summer stress effects on growth performance and meat characteristics. Another study has investigated the effects of antioxidant tomato powder to improve the oxidative stability of quail muscles under high ambient temperature (Sahin et al., 2008). Likewise, the strong antioxidant propolis has been reported to improve production and egg quality in poultry reared under high environmental temperature (Seven, 2008). Genistein (a soy flavonoid) and hesperidin (a citrus flavonoid) are 2 naturally occurring plant compounds that belong to phytochemical subgroup flavonoids. Among natural antioxidants, flavonoids are getting noticeable attention due to their multifunctional biological activities (Middleton et al., 2000). These have been proved as strong antioxidants in several in vitro studies (Rice-Evans and Miller, 1996). In animal studies, soybean flavonoids (mixture of genistein, daidzein, and glycitein) and hesperidin have been observed as free radical scavengers and to protect the muscle membrane phospholipids from oxidative spoilage (Jiang et al., 2007; Simitzis et al., 2011). These also have been reported to improve performance, carcass traits, and meat quality in commercial broiler chickens (Payne et al., 2001; Peña et al., 2008; Kamboh and Zhu, 2013). To the best of our knowledge, the supplemental effects of flavonoids in broilers reared under persistent summer climate are largely unrevealed. Therefore, the aim of present study was to investigate the supplemental effects of genistein and hesperidin on performance and biomarkers of summer heat stress in broiler chickens. The effects of combinatorial treatments of both compounds were also investigated.

SUMMER-STRESS AMELIORATING EFFECTS OF FLAVONOIDS

Hematological Analysis

2413

Analysis for Biomarkers of Heat Stress

Statistical Analysis

Breast muscle samples were analyzed for the dietary effects of genistein and hesperidin on CK and LDH levels. These were analyzed by using the commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). All protocols were adapted according to manufacturer’s instructions and samples were run in a same assay to avoid interassay variability.

All results were analyzed by using ANOVA through JMP statistical package software (version 5.0.1.a, SAS Institute Inc., Cary, NC). Tukey-Kramer test was used to determine significance of differences (P < 0.05) among different dietary treatments. Results were presented as means ± SEM.

Quantification of HSP70 mRNA Using Real-Time PCR The total RNA was isolated from the breast muscle samples using the phenol/guanidine isothiocyanate method. The purity of the extracted RNA was assessed spectrophotometrically (Eppendorf Biophotometer, Hamburg, Germany) at 260 nm. Ratios of absorption (260/280 nm) for all preparations were between 1.8 and 2.0. The integrity of the RNA samples was verified via electrophoresis using a 1.4% agarose-formaldehyde gel. Purified total RNA (2 μg) was reverse transcribed by incubation at 37°C for 1 h in a 25-μL mixture consisting of 1 × RT-buffer (Promega, Madison, WI), 100 U of Moloney Murine Leukemia Virus reverse transcriptase (Promega), 8 U of RNase inhibitor (Promega), 5.3 μmol/L of random hexamer primers (TaKaRa Biotechnology, Dalian, China), and 0.8 mmol/L of dNTP (TaKaRa Biotechnology). The reaction was terminated by heating at 95°C for 5 min and quickly cooling on ice. Reverse-transcription (RT) PCR was performed in a Bio-Rad DNA Engine Peltier Thermal Cycler PTC0200 (Bio-Rad, Hercules, CA). Real-time RT-PCR was performed in Mx3000P (Stratagene, La Jolla, CA). Mock RT and no-template controls were set to monitor the possible contamination of genomic DNA both at reverse transcription and PCR. The pooled sample made by mixing an equal quantity of total RT products (cDNA) from all samples was used for optimizing PCR condition and tailoring the standard curves for each target gene. Melting curves were performed to ensure a single specific PCR product for each gene. Two microliters of 5to 20-fold dilution of RT product was used for PCR in a final volume of 25 μL containing 12.5 μL SYBR Green Real-time PCR Master Mix (Toyobo Ltd., Osaka, Japan) and 0.2 to 0.8 μM of each forward and reverse primer for HSP70 as used previously (Figueiredo et

RESULTS Growth Performance The genistein and hesperidin supplementation improved (P < 0.05) the BW gain of broilers up to 11.3% in the finisher period (22–42 d) in comparison with the control group (Table 2). Between 22 and 28 d, all supplemented groups, except GH10; between 29 and 35 d, all supplemented groups; and between 36 and 42 d, all treated groups except G5 and GH10 significantly (P < 0.05) raised the BW gain compared with the control group. The final BW was also found to improve (P < 0.05) in the H20 group, in comparison with the control group. Feed intake (FI) was found to be improved by the end of starter period (15–21 d) and in 29 to 35 d (up to 6.4 and 4.0%, respectively) in all groups except GH10, whereas it was elevated at the end of the finisher period (36–42 d; up to 4.3%) for G5, H20, and GH20 groups compared with the control group. The feed conversion ratio (FCR) was found to be modulated (P < 0.05) in the last 2 wk of the finisher period (29–42 d), with significant differences in the H20, GH5, and GH10 groups compared with the control group. However, the overall FCR and FI did not change (P > 0.05) with the dietary treatments.

H/L Ratios The dietary effects of flavonoids genistein and hesperidin on heterophil and lymphocyte counts; and H/L ratios of broiler chickens are shown in Table 3. The heterophil count was found to be decreased (P < 0.01) in all groups except GH10, in comparison with control group with maximum fall of 14.8% (G5), followed by 13.4% (GH5), 12.4% (GH20), and 11.5% (H20). However, lymphocyte numbers were increased by the dietary treatments but their level was found statistically nonsignificant (P > 0.05). A significant reduction (P


Hematological analysis was conducted to estimate the circulating levels of lymphocytes and heterophils by using an automatic hematological analyzer (ZC-980 China). Values of lymphocytes and heterophils counts were further used to calculate the heterophil-to-lymphocyte (H/L) ratio index. All measurements were taken in triplicate and done within 2 h of sample collections to ensure the accuracy of resultant values.

al., 2007). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a reference gene for normalization purposes. The following PCR protocols included initial denaturation (1 min at 95°C), then a 3-step amplification program (20 s at 95°C, 20 to 30 s at 60 to 64°C, 15 to 20 s at 72°C) repeated 45 times. The method of 2−ΔΔCt was used to analyze the realtime RT-PCR data (Livak and Schmittgen, 2001). All samples were included in the same run of RT-PCR and repeated 3 times.

2414

Kamboh et al.

Table 2. Effect of antioxidants genistein and hesperidin on performance parameters of broiler chickens1 Treatment Parameter  

160 295 339 345 375 405 1,964 146 348 539 678 790 944 3,445 0.913 1.180 1.590 1.965 2.107 2.331 1.754

± ± ± ± ± ± ±   ± ± ± ± ± ± ±   ± ± ± ± ± ± ±

1.85 2.61 4.05 2.02c 2.92d 2.43c 21b

G5  

164 288 340 357 392 408 1,994

0.98 2.98 4.15c 5.49 3.20c 4.19c 14

140 350 552 700 808 965 3,515

0.02 0.01 0.03 0.03 0.01a 0.01a 0.04

0.854 1.215 1.624 1.961 2.061 2.365 1.763

± ± ± ± ± ± ±   ± ± ± ± ± ± ±   ± ± ± ± ± ± ±

H20  

1.63 3.15 3.88 2.89ab 2.47c 3.40bc 26b

144 266 332 380 423 436 2,026

1.25 2.99 2.98ab 6.88 3.2ab 4.27ab 15

154 346 548 695 814 966 3,523

0.00 0.01 0.02 0.03 0.02ab 0.02a 0.05

1.069 1.301 1.651 1.829 1.924 2.216 1.739

± ± ± ± ± ± ±   ± ± ± ± ± ± ±   ± ± ± ± ± ± ±

GH5

2.05 2.77 3.65 3.12a 3.02a 2.45a 13a

 

161 290 340 367 399 417 2,019

1.48 3.00 2.72ab 6.34 4.85ab 5.05ab 16

136 337 576 636 807 950 3,442

0.01 0.02 0.02 0.02 0.01c 0.01b 0.04

0.845 1.162 1.694 1.733 2.023 2.278 1.705

± ± ± ± ± ± ±   ± ± ± ± ± ± ±   ± ± ± ± ± ± ±

1.83 2.05 3.76 2.99ab 2.07b 2.19b 28ab

GH10  

152 289 333 347 390 406 1,962

1.80 3.05 3.04a 5.94 3.00ab 4.67c 14

131 345 538 640 768 902 3,324

0.03 0.01 0.02 0.05 0.02c 0.01b 0.03

0.862 1.194 1.616 1.844 1.969 2.222 1.694

± ± ± ± ± ± ±   ± ± ± ± ± ± ±   ± ± ± ± ± ± ±

1.96 2.65 3.43 2.77c 2.76c 2.20bc 20b

GH20  

159 287 337 350 400 412 1,990

1.15 2.88 3.17c 5.86 3.19bc 3.80cd 14

142 338 545 682 823 987 3,517

0.02 0.02 0.01 0.02 0.01c 0.01b 0.03

0.893 1.178 1.617 1.949 2.058 2.396 1.767

± ± ± ± ± ± ±   ± ± ± ± ± ± ±   ± ± ± ± ± ± ±

1.86 2.73 3.46 2.86ab 2.16b 2.05b 15b 1.04 3.08 2.12ab 6.02 3.22a 3.09a 12 0.00 0.01 0.03 0.02 0.01ab 0.02a 0.06

a–dMeans

that do not share common letters differ significantly (P < 0.05). value represents the mean ± SEM. BWG = BW gain; FI = feed intake; FCR = feed conversion ratio. 1–21 d = starter period; 22–42 d = finisher period. The control group was fed no additive. Other groups were fed 5 mg∙kg−1 of genistein (G5); or 20 mg∙kg−1 of hesperidin (H20); or a mixture of genistein and hesperidin (1:4) in doses of 5 mg∙kg−1 (GH5); 10 mg∙kg−1 (GH10), and 20 mg∙kg−1 (GH20), respectively. 1Each

< 0.01) was recorded in H/L ratio for all supplemental groups as compared with the control group. This decrease in H/L ratio by the dietary treatments was recorded as G5 (24.6%), H20 (21.5%), GH5 (18.1%), GH10 (20.8%), and GH20 (23.0%) groups.

with the control group (Figure 1B). Overall, the effects of combined supplementation were dominant for both CK and LDH activity in comparison with individual treatments (Figure 1A, 2B).

HSP70 mRNA Gene Expression

Biomarkers of Heat Stress It was noted that all genistein- and hesperidin-supplemented groups significantly decreased (P < 0.01) the CK activity in breast muscle (Figure 1A) in comparison with the control group. This reduction was recorded in a dose-dependent manner for combined supplemented groups as 20.9, 21.9, and 29.4% in GH5, GH10, and GH20 groups, respectively. The LDH activity was also found decreased by the dietary flavonoids with a significant difference (P < 0.01) in G5 (17.4%), GH5 (18.9%), GH10 (27.6%), and GH20 (30%) groups, in comparison

The relative values for HSP70 mRNA expression in chicken breast muscle have been shown in Figure 2. Real-time PCR analysis showed that dietary genistein and hesperidin downregulated the HSP70 gene expression with a significant difference (P < 0.01) in G5, GH10, and GH20 groups compared with the control group.

DISCUSSION Several studies have demonstrated the effects of experimental heat stress including cycling heat stress,

Table 3. Effects of antioxidants genistein and hesperidin on heterophil and lymphocyte concentrations, and heterophil-to-lymphocyte (H/L) ratio of broiler chickens1 Treatment Parameter Lymphocytes (L; × 109/L) Heterophils (H; × 109/L) H/L ratio a,bMeans

Control

G5

H20

GH5

GH10

GH20

103.50 ± 1.65 35.77 ± 0.72a 0.35 ± 0.01a

116.86 ± 1.18 30.48 ± 0.53b 0.26 ± 0.00b

116.67 ± 2.24 31.65 ± 0.51b 0.27 ± 0.01b

111.37 ± 4.25 30.98 ± 1.25b 0.28 ± 0.02b

118.38 ± 2.84 32.28 ± 1.18ab 0.27 ± 0.01b

117.60 ± 1.29 31.32 ± 0.51b 0.26 ± 0.00b

that do not share common letters differ significantly (P < 0.05). value represents the mean ± SEM; n = 12. The control group was fed no additive. Other groups were fed 5 mg∙kg−1 of genistein (G5); or 20 mg∙kg−1 of hesperidin (H20); or a mixture of genistein and hesperidin (1:4) in doses of 5 mg∙kg−1 (GH5); 10 mg∙kg−1 (GH10), and 20 mg∙kg−1 (GH20), respectively. 1Each


BWG (g) 0–7 d 8–14 d 15–21 d 22–28 d 29–35 d 36–42 d Final BW (g) FI (g) 0–7 d 8–14 d 15–21 d 22–28 d 29–35 d 36–42 d Overall FCR 0–7 d 8–14 d 15–21 d 22–28 d 29–35 d 36–42 d Overall

Control


2415


Figure 1. Effects of antioxidants genistein and hesperidin on (A) creatine kinase (expressed as U∙mg−1 of protein) and (B) lactate dehydrogenase (expressed as U∙g−1 of protein) levels of chicken breast muscle. Means (n = 12) are accompanied by SEM bars. Means that do not share common letters (a–d) differ significantly (P < 0.05). CON = control group fed no additive. Other groups were fed 5 mg∙kg−1 of genistein (G5); or 20 mg∙kg−1 of hesperidin (H20); or a mixture of genistein and hesperidin (1:4) in doses of 5 mg∙kg−1 (GH5); 10 mg∙kg−1 (GH10), and 20 mg∙kg−1 (GH20), respectively.

short-term heat stress, long-term heat stress, and acute heat stress in farm animals and have recognized the degree of heat stress-induced impairments through biomarkers including malondialdehyde, HSP, aspartate transaminase, LDH, CK, corticosterones, thyroid hormones, and circulating H/L levels (Chiang et al., 2008;

Konca et al., 2009; Cīrule et al., 2012). Nevertheless, the effects of persistent seasonal heat stress in broiler chickens were not known and there was no information available on the possible management through dietary antioxidants (phytochemicals). Therefore, keeping in view the gravity of persistent seasonal heat stress ef-

2416

Kamboh et al.

fects in broiler production, we investigated for the first time the effects of antioxidant flavonoids genistein and hesperidin for summer heat stress in broiler chickens. Our results demonstrated that antioxidants genistein and hesperidin improved the performance of broilers particularly in the finisher stage. The low effects of dietary compounds during starter period might be due to the high temperature requirements of birds in the brooding/starter stage (e.g., 33–34°C in the first week). It is generally regarded that heat stress in poultry begins to occur when temperature rises above 27°C, with a decreased FI and weight gain (Kutlu, 2001). A previous study has also reported the improving effect of ascorbic acid for performance in the finisher period of broiler chickens (Konca et al., 2009). The overall FI and FCR for the entire growth period (0–42 d) were not affected by the dietary treatments of genistein and hesperidin. This observation is in agreement with a previous study dealing with combined supplementation of ascorbic acid and citric flavonoids to female broilers under cyclic heat stress (Peña et al., 2008). Previous studies have established that H/L ratio is a reliable stress estimator in birds (Vleck et al., 2000) and known to rise due to the long lasting environmental stress factors including heat stress (Mashaly et al., 2004). Our results of H/L ratio in the present study have demonstrated that dietary genistein and hesperidin could potentially ameliorate the sustained seasonal heat stress. In addition to H/L ratio, the magnitude of leukocyte change (decreased heterophil count) is also a positive indication of ameliorating action of dietary antioxidants against heat stress, as indicated in recent studies (Oyagbemi and Adejinmi, 2012). Circu-

lating leukocytes are involved in immunological functions with pronounced phagocytic role of heterophils. In the animal body, under stress conditions, glucocorticoids caused the rapid influx of heterophils into the blood from bone marrow that increased the concentration of circulating heterophils. Nevertheless, H/L ratio is known as more important indicator for long-term stresses than assessing corticosterone levels (Cīrule et al., 2012). In birds, a rapid change in H/L ratio (upon 1 h exposure of stress) and gradual decline with continuity of stress have been reported (López-Olvera et al., 2007) that reflect the involvement of highly dynamic immunological response with quick and long-lasting effects. In the present study, the magnitude of leukocyte change (i.e., decreased heterophils and H/L ratio, and increased lymphocyte level in supplemented groups compared with unsupplemented groups) possibly indicates the anti-heat stress potential of phytochemicals. It is generally recognized that in the absence of any induced muscle damage (e.g., mechanical or biochemical), normal circulating levels of CK are the consequence of muscle cell turnover, and if found elevated will obviously demonstrate myopathy (Hamburg et al., 1991). However, some studies have addressed that CK or LDH values of a particular tissue/organ are more reliable estimation of isozyme activity of that tissue (Dawson and Fine, 1967). In present study we observed the lower levels of CK and LDH in breast muscles, which probably are an indication of ameliorating effects of dietary flavonoids against persistent summer stress. This is in agreement with previous studies that used phytochemical-rich tomato powder and vitamins and concluded that dietary antioxidants are the prime


Figure 2. Effects of antioxidants genistein and hesperidin on relative values of HSP70 mRNA expression in chicken breast muscle. Means (n = 6) are accompanied by SEM bars. Means that do not share common letters (a,b) differ significantly (P < 0.05). CON = control group fed no additive. Other groups were fed 5 mg∙kg−1 of genistein (G5); or 20 mg∙kg−1 of hesperidin (H20); or a mixture of genistein and hesperidin (1:4) in doses of 5 mg∙kg−1 (GH5); 10 mg∙kg−1 (GH10), and 20 mg∙kg−1 (GH20), respectively.


afford a protective effect against environmental stresses (Rusak et al., 2002; Bongiovanni et al., 2007). In conclusion, the present study for the first time investigated the dietary effects of genistein and hesperidin in broiler chickens under persistent summer stress. Dietary flavonoids modulated the biomarkers of heat stress, including circulating H/L ratio and CK, LDH, and HSP70 mRNA levels of chicken breast muscle in a constructive direction. The dietary compounds also improved growth performance (FI, BW, and FCR). These results indicated the protective effects of dietary flavonoids against persistent summer stress effects in broilers. Marked effects of combined supplementation was observed in this study, which might indicate the potential of both compounds for mutual (synergistic) interaction that needs to be investigated further through special model studies. It could be suggested that dietary use of genistein and hesperidin as a feed supplement may offer a potential nutritional strategy in tropical and subtropical broiler farming to overcome the deleterious effects of persistent summer stress in broiler production.

ACKNOWLEDGMENTS This work was supported by Special Fund for AgroScientific Research in the Public Interest of China (200903003). The author Asghar Ali Kamboh is highly grateful to Sindh Agriculture University, Tandojam, Pakistan, and the Higher Education Commission of Pakistan for providing a scholarship for his studies.

REFERENCES Alvarez, M. A., N. B. Debattista, and N. B. Pappano. 2008. Antimicrobial activity and synergism of some substituted flavonoids. Folia Microbiol. (Praha) 53:23–28. Bongiovanni, G. A., E. A. Soria, and A. R. Eynard. 2007. Effects of the plant flavonoids silymarin and quercetin on arsenite-induced oxidative stress in CHO-K1 cells. Food Chem. Toxicol. 45:971–976. Chiang, W., A. Booren, and G. Strasburg. 2008. The effect of heat stress on thyroid hormone response and meat quality in turkeys of two genetic lines. Meat Sci. 80:615–622. Cīrule, D., T. Krama, J. Vrublevska, M. Rantala, and I. Krams. 2012. A rapid effect of handling on counts of white blood cells in a wintering passerine bird: A more practical measure of stress? J. Ornithol. 153:161–166. Daroit, D. J., and A. Brandelli. 2008. Implications of skeletal muscle creatine kinase to meat quality. J. Anim. Feed Sci. 17:285–294. Dawson, D. M., and I. H. Fine. 1967. Creatine kinase in human tissues. Arch. Neurol. 16:175–180. Figueiredo, D., A. Gertler, G. Cabello, E. Decuypere, J. Buyse, and S. Dridi. 2007. Leptin downregulates heat shock protein-70 (HSP-70) gene expression in chicken liver and hypothalamus. Cell Tissue Res. 329:91–101. Ghayour-Mobarhan, M., S. A. New, D. J. Lamb, B. L. Starkey, C. Livingstone, T. Wang, N. Vaidya, and G. A. Ferns. 2005. Dietary antioxidants and fat are associated with plasma antibody titers to heat shock proteins 60, 65, and 70 in subjects with dyslipidemia. Am. J. Clin. Nutr. 81:998–1004. Hamburg, R. J., D. L. Friedman, and M. B. Perryman. 1991. Metabolic and diagnostic significance of creatine kinase isoenzymes. Trends Cardiovasc. Med. 1:195–200.


strategy against the undesirable consequences of heat stress in poultry meat (Sahin et al., 2008; Patra et al., 2011). A recent study on rats demonstrated a protective role of hesperidin against cyclophosphamide-induced oxidative damage manifested by the downregulation of muscle creatinine phosphokinase and LDH (Kumar et al., 2011). Therefore, the results may suggest that dietary genistein and hesperidin could play a role in the reduction of myopathy that is marked by the reduced muscular activities of CK and LDH. Another interesting finding in the present study is that the effects of combined genistein and hesperidin supplementation were more dominant than their individual treatments for CK and LDH activities. These observations in recent studies had been termed “effect of combination” and are known as synergistic activities (Reber et al., 2011). However, it is well recognized that flavonoids upon combination could produce synergistic, additive, or antagonistic effects (Alvarez et al., 2008). Nevertheless, further studies are warranted to investigate this phenomenon through specially designed projects. Heat shock proteins are a well-conserved group of transcriptional activators considered as vital for cell survival in response to a variety of external stressors including thermotolerance (i.e., protection from further exposure to heat shock). One of the major reasons of growing interest of biomedical research in HSP are the evidences that support their role not only in heat stress but also in cell death mediated by free radicals and reactive oxygen species (Jacquier-Sarlin et al., 1994). Moreover, epidemiological studies and animal models have also reported the inverse association between dietary antioxidants and HSP expression (Ghayour-Mobarhan et al., 2005). In the present study, the downregulation of HSP70 mRNA by the G5 and combined supplemented groups (GH10 and GH20) might be due to structural and functional resemblance of genistein with estrogen. Indeed, previous studies have addressed that 17β-estradiol could attenuate the plasma CK activity and HSP70 expression in skeletal muscles in a non-receptor-mediated way (Paroo et al., 2002). Our novel finding suggested a mechanism of action for genistein similar to that of endogenous estrogen for HSP70 induction through a nongenomic hormonal mechanism (Setchell, 2001). Other studies have demonstrated the molecular mechanism for stress response suppression whereby genistein exerts its inhibition through interference of a key transcription factor, CBF/NF-Y, binding to the most proximal CCAAT site of the HSP70 and GRP78 promoters (Zhou and Lee, 1998). Our present results are in agreement with a study of Japanese quail that reported the genistein for downregulation of muscular HSP70 and HSP60 levels (Sahin et al., 2009). In vitro studies dealing with flavonoids have also reported the pronounced effects of these natural antioxidants against acute and chronic stressors including heat stress and concluded that dietary inclusion of flavonoids may

2417

2418

Kamboh et al. Patra, T., P. K. Pati, and A. K. Mohapatra. 2011. Study on carcass quality of coloured broiler chicks supplemented with vitamin E and C during summer stress. SAARC J. Agric. 9:123–132. Payne, R. L., T. D. Bidner, L. L. Southern, and K. W. McMillin. 2001. Dietary effects of soy isoﬂavones on growth and carcass traits of commercial broilers. Poult. Sci. 80:1201–1207. Peña, J. E. M., S. L. Vieira, J. López, R. N. Reis, R. Barros, F. V. F. Furtado, and P. X. Silva. 2008. Ascorbic acid and citric flavonoids for broilers under heat stress: Effects on performance and meat quality. Braz. J. Poult. Sci. 10:125–130. Poels, P. J. E., and F. J. M. Gabreëls. 1993. Rhabdomyolysis: A review of the literature. Clin. Neurol. Neurosurg. 95:175–192. Reber, J. D., D. L. Eggett, and T. L. Parker. 2011. Antioxidant capacity interactions and a chemical/structural model of phenolic compounds found in strawberries. Int. J. Food Sci. Nutr. 62:445–452. Rice-Evans, C. A., and N. J. Miller. 1996. Antioxidant activities of flavonoids as bioactive components of food. Biochem. Soc. Trans. 24:790–795. Rusak, G., H. O. Gutzeit, and J. Ludwig-Müller. 2002. Effects of structurally related flavonoids on hsp gene expression in human promyeloid leukaemia cells. Food Technol. Biotechnol. 40:267– 273. Sahin, K., F. Akdemir, M. Tuzcu, N. Sahin, M. Onderci, R. Ozercan, N. Ilhan, E. Kilic, S. Seren, and O. Kucuk. 2009. Genistein suppresses spontaneous oviduct tumorigenesis in quail. Nutr. Cancer 61:799–806. Sahin, N., C. Orhan, M. Tuzcu, K. Sahin, and O. Kucuk. 2008. The effects of tomato powder supplementation on performance and lipid peroxidation in quail. Poult. Sci. 87:276–283. Setchell, K. D. R. 2001. Soy isoflavones, benefits and risks from nature’s selective estrogen receptor modulators (SERMs). J. Am. Coll. Nutr. 20:354S–362S. Seven, P. T. 2008. The effects of dietary Turkish propolis and vitamin C on performance, digestibility, egg production and egg quality in laying hens under different environmental temperatures. Asian-australas. J. Anim. Sci. 21:1164–1170. Simitzis, P. E., G. K. Symeon, M. A. Charismiadou, A. G. Ayoutanti, and S. G. Deligeorgis. 2011. The effects of dietary hesperidin supplementation on broiler performance and chicken meat characteristics. Can. J. Anim. Sci. 91:275–282. Vleck, C. M., N. Vertalino, D. Vleck, and T. L. Bucher. 2000. Stress, corticosterone and heterophil to lymphocyte ratios in free-living Adélie penguins. Condor 102:392–400. Woelfel, R. L., C. M. Owens, E. M. Hirschler, R. Martinez-Dawson, and A. R. Sams. 2002. The characterization and incidence of pale, soft, and exudative broiler meat in a commercial processing plant. Poult. Sci. 81:579–584. Zhou, Y., and A. S. Lee. 1998. Mechanism for the suppression of the mammalian stress response by genistein, an anticancer phytoestrogen from soy. J. Natl. Cancer Inst. 90:381–388.


Imik, H., M. A. Atasever, M. Koc, M. Atasever, and K. Ozturan. 2010. Effect of dietary supplementation of some antioxidants on growth performance, carcass composition and breast meat characteristics in quails reared under heat stress. Czech J. Anim. Sci. 55:209–220. Jacquier-Sarlin, M. R., K. Fuller, A. T. Dinh-Xuan, M. J. Richard, and B. S. Polla. 1994. Protective effects of hsp70 in inflammation. Experientia 50:1031–1038. Jiang, Z. Y., S. Q. Jiang, Y. C. Lin, P. B. Xi, D. Q. Yu, and T. X. Wu. 2007. Effects of soybean isoflavone on growth performance, meat quality, and antioxidation in male broilers. Poult. Sci. 86:1356–1362. Kamboh, A. A., and W. Y. Zhu. 2013. Effect of increasing levels of bioflavonoids in broiler feed on plasma anti-oxidative potential, lipid metabolites, and fatty acid composition of meat. Poult. Sci. 92:454–461. Konca, Y., F. Kirkpinar, S. Mert, and S. Yurtseven. 2009. Effects of dietary ascorbic acid supplementation on growth performance, carcass, bone quality and blood parameters in broilers during natural summer temperature. Asian J. Anim. Vet. Adv. 4:139– 147. Kumar, S., N. Dhankhar, V. Kar, M. Shrivastava, and S. Shrivastava. 2011. Myocardial injury provoked by cyclophosphamide, protective aspect of hesperidin in rats. Int. J. Res. Pharm. Biomed. Sci. 2:1288–1296. Kutlu, H. R. 2001. Influence of wet feeding and supplementation with ascorbic acid on performance and carcass composition of broiler chicks exposed to a high ambient temperature. Arch. Tierernahr. 54:127–139. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. López-Olvera, J. R., I. Marco, J. Montané, E. Casas-Díaz, and S. Lavín. 2007. Effects of acepromazine on the stress response in southern chamois (Rupicapra pyrenaica) captured by means of drive-nets. Can. J. Vet. Res. 71:41–51. Mashaly, M. M., G. L. Hendricks 3rd, M. A. Kalama, A. E. Gehad, A. O. Abbas, and P. H. Patterson.2004. Effect of heat stress on production parameters and immune responses of commercial laying hens. Poult. Sci. 83:889–894. McCurdy, R. D., S. Barbut, and M. Quinton. 1996. Seasonal effect on pale soft exudative (PSE) occurrence in young turkey breast meat. Food Res. Int. 29:363–366. Middleton, E., C. Kandaswami, and T. Theoharides. 2000. The effect of plant flavonoids on mammalian cells: Implications for inflammation, heart disease and cancer. Pharmacol. Rev. 52:673–751. NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Oyagbemi, T. O., and J. O. Adejinmi. 2012. Supplementation of broiler feed with leaves of Vernonia amygdalina and Azadirachta indica protected birds naturally infected with Eimeria sp. Afr. J. Biotechnol. 11:8407–8413. Paroo, Z., E. S. Dipchand, and E. G. Noble. 2002. Estrogen attenuates postexercise HSP70 expression in skeletal muscle. Am. J. Physiol. Cell Physiol. 282:C245–C251.