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chickens with the increase in ambient temperature. There are several reports (Deyhim and Teeter, 1991;. Berrong and Washburn, 1998; Cooper and Washburn,.
Effects of different acute high ambient temperatures on function of hepatic mitochondrial respiration, antioxidative enzymes, and oxidative injury in broiler chickens G.-Y. Tan,*† L. Yang,† Y.-Q. Fu,*† J.-H. Feng,* and M.-H. Zhang*1 *State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P. R. China; and †Department of Animal Nutrition and Feed Science, College of Animal Science, South China Agricultural University, Guangzhou 510642, P. R. China ABSTRACT This study investigated the effects of different acute high ambient temperatures on dysfunction of hepatic mitochondrial respiration, the antioxidative enzyme system, and oxidative injury in broiler chickens. One hundred twenty-eight 6-wk-old broiler chickens were assigned randomly to 4 groups and subsequently exposed to 25 (control), 32, 35, and 38°C (RH, 70 ± 5%) for 3 h, respectively. The rectal temperatures, activity of antioxidative enzymes (superoxide dismutase, catalase, and glutathione peroxidase), content of malondialdehyde and protein carbonyl, and the activity of mitochondrial respiratory enzymes were determined. The results showed that exposure to high ambient temperature induced a significant elevation of rectal temperature, antioxidative enzyme activity, and formation of malondialdehyde and protein carbonyl, as well as dysfunction of the mitochondrial respiratory chain in comparison with control (P < 0.05). Almost all of the

indicators changed in a temperature-dependent manner with the gradual increase of ambient temperature from 32 to 38°C; differences in each parameter (except catalase) among the groups exposed to different high ambient temperatures were also statistically significant (P < 0.05). The results of the present study suggest that, in the broiler chicken model used here, acute exposure to high temperatures may depress the activity of the mitochondrial respiratory chain. This inactivation results subsequently in overproduction of reactive oxygen species, which ultimately results in oxidative injury. However, this hypothesis needs to be evaluated more rigorously in future studies. It has also been shown that, with the gradual increase in temperature, the oxidative injury induced by heat stress in broiler chickens becomes increasingly severe, and this stress response presents in a temperature-dependent manner in the temperature range of 32 to 38°C.

Key words: high ambient temperature, reactive oxygen species, mitochondria respiration complex, oxidative injury, broiler 2010 Poultry Science 89:115–122 doi:10.3382/ps.2009-00318

INTRODUCTION

ambient temperatures has been demonstrated in several studies (Altan et al., 2003; Mujahid et al., 2005, 2006, 2007b; Lin et al., 2006, 2008a). The results from previous studies suggest that different breeds and strains of chickens may have different susceptibility to heat stress induced by high ambient temperatures (Washburn et al., 1980; Altan et al., 2003). Moreover, the resistance to heat stress of the strains selected for rapid growth is significantly less than that of a slow-growing strain (Washburn et al., 1980), and continuous selection for fast growth has been associated with increased susceptibility of broilers to heat stress (Washburn et al., 1980; Cahaner et al., 1995; Berrong and Washburn, 1998). The Arbor Acres broiler chicken, an example of a meat-type broiler breed that shows rapid growth, is commonly used because it is a readily obtainable commercial breed. However, to date, there have been few reports on the oxidative in-

Heat stress is one of the most important stressors associated with economic losses to the poultry industry in the hotter regions of the world. It causes poor growth performance (Bottje and Harrison, 1985), immunosuppression (Young, 1990), and high mortality (Yahav et al., 1995). It is now known that heat stress can enhance the formation of reactive oxygen species (ROS), which can cause oxidative injury such as lipid peroxidation and oxidative damage to proteins and DNA (Halliwell and Aruoma, 1991; Flanagan et al., 1998; Lord-Fontaine and Averill-Bates, 2002; Mujahid et al., 2007b). In broiler chickens, the oxidative injury induced by high ©2010 Poultry Science Association Inc. Received July 2, 2009. Accepted September 6, 2009. 1 Corresponding author: [email protected]

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jury induced by heat stress in this breed when exposed acutely to different high ambient temperatures. It is also known that the primary source of ROS is leakage of electrons from the respiratory chain during the reduction of molecular oxygen to water, which generates the superoxide anion (Boveris et al., 1972). However, the mechanism of ROS production in chickens subjected to heat stress still requires elucidation. Mujahid et al. speculated that the mechanisms through which increased ROS production occurs in heat-treated meat-type chickens may be associated with mitochondrial damage, such as a reduction in the activity of the mitochondrial respiratory chain complex (Mujahid et al., 2005), or a downregulation of the synthesis of avian uncoupling protein (Mujahid et al., 2005, 2006). Subsequent studies have shown that overproduction of mitochondrial ROS in chicken skeletal muscle under conditions of heat stress may result from enhanced substrate oxidation and downregulation of avian uncoupling protein in a time-dependent manner (Mujahid et al., 2007a). However, there have been few reports on the function of the mitochondrial respiratory chain in broiler chickens exposed to heat stress. In view of these considerations, the objective of the present study was to determine the effects of acute exposure to different high ambient temperatures on the activity of the mitochondrial respiratory chain complex, modification of the antioxidative enzyme system, and oxidative injury. The findings of the present study also provide further biological evidence in support of the notion that high ambient temperature can cause oxidative injury in meat-type chickens.

MATERIALS AND METHODS Birds and Experimental Design Arbor Acres broiler chickens, purchased from Beijing Huadu Poultry Breeding Co. Ltd. (Beijing, China), were reared in electrically heated battery cages from d 1, following the procedures described in the Arbor Acres Broilers Husbandry Manual. One hundred twenty-eight 6-wk-old broiler chickens were divided randomly into 4 groups. Each group consisted of 8 replicates, and each replicate was a cage containing 4 birds. The birds were housed in a controlled-environment chamber at 25°C and 70 ± 5% RH for 6 d. After the adaptation period, the broilers were exposed to conditions of various high ambient temperatures (32, 35, and 38°C; RH = 70 ± 5%; for 3 h) and their behavioral response was observed. For the control group, the temperature was 25°C and RH was 70 ± 5%. Feed and water were offered for ad libitum consumption.

Measurement of Rectal Temperature and Sampling Preparation At the end of each experiment, the rectal temperatures of 3 chickens in each replicate were determined

with a thermocouple probe before the chickens were killed. For the rectal temperature measurements, a thermocouple was inserted 4 cm into the rectum for 30 s. Samples of blood and liver were obtained after heat exposure. The serum was isolated by centrifugation for 10 min at 2,500 × g and stored at −80°C for analysis. The liver samples were harvested and immediately frozen in liquid nitrogen and stored at −80°C until subsequent analysis.

Antioxidative Enzyme Activity and Lipid Peroxidation Oxidative stress results from an imbalance between free radical generation and antioxidant defense systems (Ames et al., 1993; Sandhu and Kaur, 2002). Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSHPx) are the first line defense antioxidants (Ray and Husain, 2002). Malondialdehyde (MDA) is the main oxidation product of peroxidized polyunsaturated fatty acids and increased MDA level is an important indication of lipid peroxidation. In the present study, the activities of SOD, CAT, GSH-Px, and MDA in liver and serum were determined (TU-1810, Pgeneral, Beijing, China) by the corresponding assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China; Tan et al., 2008) according to the instructions of the manufacturer. The protein concentrations in liver tissue were determined using the Coomassie Brilliant Blue G-250 reagent with BSA as a standard.

Measurement of Protein Carbonyl Formation Protein oxidation has been associated with various diseases and oxidative stress (Ge et al., 2009). Protein carbonyl, ketone, and aldehyde groups are acceptable as biomarkers for protein damage. This study evaluated protein carbonyl content in the liver tissue homogenate by the method based on the reaction of carbonyl groups with 2, 4-dinitrophenylhydrazine to form 2, 4-dinitrophenylhydrazone (Lenz et al., 1989; Levine et al., 1990).

Isolation of Hepatic Mitochondria Hepatic mitochondria were isolated by differential centrifugation as described by previous studies (Rustin et al., 1994; Cawthon et al., 1999), with modifications. Briefly, 1 g of liver was placed in 10 mL of isolation medium (210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 0.2 mg/mL of BSA, and 1 mM ethylene glycol tetraacetic acid, pH 7.4), minced, and homogenized with a tissue grinder (10,000 cycles/min, twice for 15 s). The homogenate was then centrifuged twice for 10 min at 1,300 × g, and the supernatant was centrifuged again at 8,700 × g for 10 min. The final mitochondrial pellet was resuspended in medium containing 210 mM mannitol,

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70 mM sucrose, and 5 mM HEPES, pH 7.4. Mitochondrial protein concentrations were determined using the Coomassie Brilliant Blue G-250 reagent (27816, Fluka, Buchs, Switzerland) with BSA as a standard.

data were expressed as means ± SEM setting P < 0.05 as a criterion of statistical significance.

Assays for Activity of Mitochondrial Respiratory Enzymes

The characteristic behavioral responses of the chickens under conditions of different ambient temperatures were observed during the experiments. The chickens in the control group showed normal behaviors, whereas in the groups exposed to high ambient temperatures, the chickens showed increasingly significant abnormal behavioral responses (e.g., panting, spread wings, fluffedup feathers, drinking more water, depression, watery stool) as the temperature was increased from 32 to 38°C. This observation indicates that with the gradual increase in ambient temperature, the heat stress became more and more severe. The effect of acute exposure to high ambient temperature on rectal temperature is shown in Figure 1. After 3 h of exposure, high ambient temperatures (32, 35, and 38°C; 70 ± 5%) induced significant elevation of rectal temperature in comparison with control values (25°C, 70 ± 5%). In the groups exposed to high ambient temperatures, the rectal temperature rose in a temperature-dependent manner, and there were significant differences among all of the treatment groups (P < 0.05). The activities of SOD, GSH-Px, and CAT are shown in Figure 2, Figure 3, and Figure 4, respectively. After 3 h of exposure, there were significant differences in the activities of SOD and GSH-Px (P < 0.05) in serum and liver between the control and the groups exposed to high ambient temperature. In the latter groups, the activity of SOD and GSH-Px rose in a temperaturedependent manner, and there were significant differences among all of the treatments groups (Figures 2

The mitochondrial respiratory enzymes assay was carried out according to previous studies (Chuang et al., 2002; Chan et al., 2005). All enzyme assays were performed using a UV-visible spectrophotometer (TU1810, Pgeneral). At least duplicate determination was carried out for each tissue sample in all enzyme assays. All reagents used in enzyme assays were purchased from one supplier (Sigma-Aldrich Corp., St .Louis, MO). Nicotinamide adenine dinucleotide cytochrome c reductase (NCCR; complexes I + III) activity was determined by the reduction of oxidized cytochrome c measured at 550 nm and was calculated as the difference in the presence or absence of rotenone. The activity was assayed in 50 mM K2HPO4 buffer (pH 7.4) containing 1.5 mM KCN, 1.0 mM NADH, and 5 μL of mitochondrial suspension in the presence or absence of rotenone (20 μM). The reaction was initiated after 2 min of stabilization by adding 0.1 mM cytochrome c, and absorbance at 550 nm was measured at 5-s intervals over the first 3 min at 37°C. The molar extinction coefficient of cytochrome c at 550 nm is 28 mM−1·cm−1. Determination of succinate cytochrome c reductase (SCCR; complexes II + III) activity was performed in 40 mM K2HPO4 buffer (pH 7.4) containing 20 mM succinate, 1.5 mM KCN, and 5 μL of mitochondrial suspension. After 5 min of incubation at 37°C, the reaction was initiated by adding 50 μM cytochrome c, and absorbance at 550 nm was measured at 5-s intervals over the first 2 min at 37°C. Cytochrome c oxidase (CCO; complex IV) activity was measured by recording the oxidation of reduced cytochrome c at 550 nm. The activity of CCO is defined as the first order rate constant and is calculated from the known concentration of ferrocytochrome c and the amount of enzyme in the assay mixture. The activity was assayed in 10 mM K2HPO4 buffer (pH 7.4) containing 1.5 mM KCN, 20 mM succinate, and 5 μL of mitochondrial suspension. After 5 min of incubation at 30°C, the reaction was initiated by adding 45 μM ferrocytochrome c, and absorbance at 550 nm was measured at 5-s intervals over the first 3 min.

RESULTS

Statistical Analysis Statistical analyses were carried out using the SPSS version 17.0 software (SPSS Inc., Chicago, IL) and figures were generated by using SigmaPlot version 10.0 (Systat Software Inc., San Jose, CA). Data for all of the groups of birds were compared using 1-way ANOVA followed by Duncan’s multiple comparison tests. The

Figure 1. Effect of acute exposure to high ambient temperature on rectal temperature. Values are mean ± SEM of 8 replications. Data points with different letters are significantly different (1-way ANOVA) at the level of P < 0.05 by Duncan’s multiple comparison test.

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and 3). The activity of CAT in the liver and serum also showed a temperature-dependent increasing trend, although only the groups exposed to the highest ambient temperature (38°C) showed a significant difference in comparison with control (Figure 4). These results demonstrate that exposure to high ambient temperature can cause a compensatory increase in the activity of antioxidative enzymes. The content of protein carbonyl and MDA is shown in Figures 5 and 6, respectively. In comparison with the controls, high ambient temperature induced a significant upregulation of MDA production (P < 0.05) and protein carbonyl formation (P < 0.05). These 2 indicators both showed a temperature-dependent increasing trend, and the differences among the groups exposed to each high ambient temperature were also statically significant (P < 0.05). Thus, exposure to high ambient temperature can cause temperature-dependent oxidative injury in lipids and proteins.

The changes in mitochondrial function in liver tissue after exposure to different ambient temperatures were evaluated by examination of the activity of key enzymes in respiratory complexes I + III (NCCR), II + III (SCCR), and IV (CCO). The activities of NCCR, SCCR, and CCO in hepatic mitochondria are shown in Figure 7. In comparison with control, it can be seen that although the activity of NCCR and CCO in hepatic mitochondria underwent a significant decrease during the exposure to high ambient temperature (P < 0.05), SCCR remained essentially unchanged (P > 0.05). Gradual increase in ambient temperature from 32 to 38°C induced a gradual decrease in NCCR and CCO enzyme activity in response to heat stress, with significant differences between groups exposed to each high temperature.

Figure 2. Effect of acute exposure to high ambient temperature on the activity of superoxide dismutase (SOD) in liver and serum. Values are mean ± SEM of 8 replications. Data points with different letters are significantly different (1-way ANOVA) at the level of P < 0.05 by Duncan’s multiple comparison test.

Figure 3. Effect of acute exposure to high ambient temperature on the activity of glutathione peroxidase (GSH-Px) in liver and serum. Values are mean ± SEM of 8 replications. Data points with different letters are significantly different (1-way ANOVA) at the level of P < 0.05 by Duncan’s multiple comparison test.

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DISCUSSION In southern China and most other tropical and warm temperate regions, the average summer temperature is around 25°C, the summer maximum temperature ranges between 32 and 40°C, and the RH range is 70 to 85% (http://cdc.cma.gov.cn/). Such a high ambient temperature increases the likelihood of heat stress in poultry. According to the recommendations of the Broiler Commercial Management Guide, the correct environmental temperature range for 6-wk-old broiler chickens is 21 to 25°C. Based on these data, in our experiment, the control group was housed in a controlled-environment chamber at 25°C (RH, 70 ± 5%), whereas the high ambient temperature groups were maintained at 32, 35, and 38°C (RH, 70 ± 5%), respectively, for 3 h. The rectum and thorax are usually considered to be the sites from which to record body temperature (Poole and Stephenson, 1977). In the present study, after 3 h of exposure, the high ambient temperatures caused

Figure 4. Effect of acute exposure to high ambient temperature on the activity of catalase (CAT) in liver and serum. Values are mean ± SEM of 8 replications. Data points with different letters are significantly different (1-way ANOVA) at the level of P < 0.05 by Duncan’s multiple comparison test.

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significantly higher rectal temperatures in the broiler chickens with the increase in ambient temperature. There are several reports (Deyhim and Teeter, 1991; Berrong and Washburn, 1998; Cooper and Washburn, 1998) that suggest that heat stress results in an increase in body temperature. Additionally, the abnormal characteristic responses observed are consistent with an increase in rectal temperature in broiler chickens. This observation indicates that the high ambient temperatures caused significant heat stress, and with the gradual increase in ambient temperature, the heat stress became increasingly severe. The antioxidative enzyme system (comprising SOD, GSH-Px, and CAT) acts as the first line of antioxidant defense; modification of the activity of these enzymes can alter the balance between the production of ROS and the antioxidant system. In the present study, with an increase in ambient temperature, the activities of the main antioxidative enzymes in serum and liver were upregulated significantly. McArdle and Jackson also demonstrated a significant increase in the production of free radicals together with an increase in the expression of antioxidant enzymes during a period of nondamaging exercise (McArdle and Jackson, 2000). These increases in antioxidant enzyme activities have been considered to be a protective response against oxidative stress (Mates et al., 1999; Devi et al., 2000; Thomas, 2000). Thus, it is implied that the balance has already been disturbed by acute heat stress. This study also involved measurement of the MDA content (liver and serum) and the formation of carbonyl protein (liver) to investigate whether the superfluous ROS induced by acute heat stress caused further oxidative injury because MDA and protein carbonyl are biomarkers of lipid peroxidation (Mujahid et al., 2007b; Lin et al., 2008b) and oxidative damage of proteins (Lenz

Figure 5. Effect of acute exposure to high ambient temperature on protein carbonyl formation in liver. Values are mean ± SEM of 8 replications. Data points with different letters are significantly different (1-way ANOVA) at the level of P < 0.05 by Duncan’s multiple comparison test.

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et al., 1989; Levine et al., 1990), respectively. Moreover, the relationship in biological systems between lipid peroxidation, oxidative damage to proteins, and high temperature has been discussed previously (Borisiuk and Zinchuk, 1995; Iwagami, 1996; Carvalho et al., 1997). In the present study, chickens in the high temperature groups showed significant increases in both MDA content and protein carbonyl formation, and the difference in the level of each indicator between the groups exposed to higher and lower ambient temperatures was also significant. Thus, these observations indicate that exposure to high ambient temperatures induced oxidative damage to lipids and proteins, and such damage occurred in a temperature-dependent manner. Given that the rates of many chemical and biochemical reactions increase with temperature, it is thus very likely that elevated body temperature would enhance the generation of ROS via accelerated metabolic reactions in cells and tissues (Lin et al., 2006). Mujahid et

Figure 6. Effect of acute exposure to high ambient temperature on the level of malondialdehyde (MDA) in liver and serum. Values are mean ± SEM of 8 replications. Data points with different letters are significantly different (1-way ANOVA) at the level of P < 0.05 by Duncan’s multiple comparison test.

Figure 7. The activities of nicotinamide adenine dinucleotide cytochrome c reductase (NCCR), succinate cytochrome c reductase (SCCR), and cytochrome c oxidase (CCO) in hepatic mitochondria under different high temperature conditions. Values are mean ± SEM of 8 replications. Data points with different letters are significantly different (1-way ANOVA) at the level of P < 0.05 by Duncan’s multiple comparison test. Cyto c = cytochrome c.

al. first reported evidence of ROS production in the skeletal muscle mitochondria of meat-type chickens in response to acute heat stress (Mujahid et al., 2005), and subsequent experiments have confirmed this result (Lin et al., 2006; Feng et al., 2008). It is known that the electron transport chain in the mitochondria is the main source of cellular ROS (Boveris et al., 1972) and that strong inhibition of the activity of the respiratory chain complex can compromise adenosine triphosphate synthesis (Letellier et al., 1994; Davey and Clark, 1996; Rossignol et al., 1999), which will lead to further formation of superoxide anion radicals (Boveris et al., 1972; Liu et al., 2002; Kudin et al., 2005; Szabo et al., 2007; Murphy, 2009). In the current study, after acute exposure to different high ambient temperatures, the activities of the respiratory chain complex (NCCR and CCO, not including SCCR) were inhibited significantly in a temperature-dependent manner over the range 32 to 38°C. Previous studies have reported similar results in the rostral ventrolateral medulla of rats during endotoxemia (Chuang et al., 2002; Chan et al., 2005). Nevertheless, the reason why acute heat stress does not alter the function of complexes II + III deserves further investigation. Given this observation, the modification of the antioxidative enzyme system, and the oxidative injury induced by heat stress, we have a good reason to suspect that in the chicken model, the ROS production induced by acute heat stress was probably related to inactivation of the respiratory chain complex, although ROS production induced by heat stress was not measured directly in the current study. Taken together, the results obtained using the present broiler chicken model show that acute exposure to

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high temperatures may depress the activity of the mitochondrial respiratory chain. This inactivation results in overproduction of ROS, which ultimately results in oxidative injury. However, this hypothesis needs to be evaluated rigorously in future studies. It has also been shown that, with a gradual increase in temperature, the oxidative injury induced by heat stress in broiler chickens becomes increasingly serious, and the stress response occurs in a temperature-dependent manner in the temperature range of 32 to 38°C.

ACKNOWLEDGMENTS This study was supported by National Basic Research Program of China (Grant No. 2004CB117507). We also acknowledge the financial support provided by the program sponsored by the State Key Laboratory of Animal Nutrition [2004DA125184 (Group) 0807]. In addition, we thank the editor and anonymous reviewers.

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