METABOLISM AND NUTRITION Effects of low ambient temperatures and dietary vitamin C supplement on growth performance, blood parameters, and antioxidant capacity of 21-day-old broilers X. Yang, Y. H. Luo, Q. F. Zeng,1 K. Y. Zhang, X. M. Ding, S. P. Bai, and J. P. Wang Institute of Animal Nutrition, Key Laboratory for Animal Disease Resistance Nutrition of the Ministry of Education, Sichuan Agricultural University, Sichuan, Ya’an 625014, China
Key words: low ambient temperature, vitamin C, ascites, blood parameter, antioxidant capacity 2014 Poultry Science 93:898–905 http://dx.doi.org/10.3382/ps.2013-03438 temperature by 1°C per day from 5 to 22 d resulted in broilers experiencing right ventricular hypertrophy and death from AS. In another study, keeping ambient temperatures at 32 and 30°C during 1 and 2 wk, respectively, and then reducing it to 15°C during the third week, also led to more AS incidences in broilers (Daneshyar et al., 2009). Because of the significant temperature changes in the winter in China, the expensive thermal equipment to mitigate temperature effects, and the high incidence of AS affecting broilers in winter, it is important to study the effects of natural temperatures on the incidence of AS in winter. Previous studies have indicated that a lack of oxygen in the body of modern broilers, due the imbalance between rapid growth and limited cardiopulmonary development, is a major factor in developing AS (Julian et al., 1987; Nain et al., 2009). Nain et al. (2008) reported that fast-growing commercial broilers had a high risk of heart failure coupled with increased oxidative stress resulting from continuous hypoxia; oxidative stress caused damage to tissues and exhausted the broilers’ antioxidant supply. Previously, Enkvetchakul
INTRODUCTION Fast-growing broilers are susceptible to ascites syndrome (AS), which causes fluid accumulation in their abdominal cavities and is associated with pathological damage to the heart, liver, and lungs. This syndrome leads to increased mortality and results in substantial economic losses (Maxwell and Robertson, 1997). Hypobaric chambers (Owen et al., 1990; Julian and Squires, 1994), low ventilation (Enkvetchakul et al., 1993), and the compound 3,3c,5-triiodothyronine (Decuypere et al., 1994) have all been found to induce AS. Low ambient temperatures (LAT), which affect most farming areas in the winter, have also been reported to induce widespread incidences of AS. In a study by Julian et al. (1989), holding meat-type chickens at an ambient temperature of 30°C and then reducing the
©2014 Poultry Science Association Inc. Received June 22, 2013. Accepted December 16, 2013. 1 Corresponding author: [email protected]
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whole experimental period (P < 0.01), whereas it increased heart index at 21 d (P < 0.05) and hematocrit and hemoglobin level at 14 d (P < 0.05). Supplementing the diet with VC increased hematocrit, hemoglobin, and red blood cell count at 21 d (P < 0.05). At 21 d, LAT conditions decreased total antioxidant capacity in the serum, liver, and lungs (P < 0.05), and it also increased the levels of VC in the serum and liver, the amount of protein carbonylation in liver and lungs, and the malondialdehyde level in the lungs (P < 0.05). The addition of VC tended to increase the total antioxidant capacity level in serum (P < 0.1). Low ambient temperature resulted in oxidative stress for broilers that were fed from 1 to 21 d of age, whereas no significant effect was found on the antioxidant activity by dietary VC supplementation.
ABSTRACT The study was conducted to determine the effects of low ambient temperature (LAT) and a vitamin C (VC) dietary supplement on the growth performance, blood parameters, and antioxidant capacity of 21-d-old broilers. A total of 400 one-day-old male Cobb broilers were assigned to 1 of 4 treatments as follows: 1) LAT and a basal diet; 2) LAT and a basal diet supplemented with 1,000 mg of VC/kg (LAT + VC); 3) normal ambient temperature (NAT) and a basal diet; 4) NAT and a basal diet supplemented with 1,000 mg of VC/kg (NAT + VC). All birds were fed to 21 d of age. Broilers in groups 1 and 2 were raised at 24 to 26°C during 1 to 7 d, and at 9 to 11°C during 8 to 21 d, whereas groups 3 and 4 were raised at 29 to 31°C during 1 to 7 d and at 24 to 26°C during 8 to 21 d. The LAT increased the feed conversion ratio during the
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EFFECTS OF LOW TEMPERATURES AND VITAMIN C ON BROILERS Table 1. Temperature scheme1 Indoor temperature
Temperature inside the cage
Item
0–7 d
8–21 d
0–7 d
8–21 d
LAT (°C) NAT (°C)
24–26 29–31
9–11 24–26
27–29 31–33
11–13 27–29
1LAT
= low ambient temperature, NAT = normal ambient temperature.
MATERIALS AND METHODS Birds, Experimental Design, and Diet The animal experiment was carried out in accordance with the Chinese guidelines for animal welfare and approved by the Animal Health and Care Committee of Sichuan Agricultural University. A total of 400 one-day-old male Cobb broilers were purchased from a commercial hatchery (Wenjiang Zhengda Co. Ltd., Chengdu, Sichuan province, China). They were raised in cage pens (1.5 m × 1 m × 0.5 m) at the farm of the Animal Nutrition Institute of Sichuan Agricultural
University in China. The initial BW was similar among all pens. Feed and water were provided ad libitum. The experimental treatments were arranged as a 2 × 2 factorial as follows: 1) LAT and a basal diet; 2) LAT and a basal diet supplemented with 1,000 mg of VC/kg (LAT + VC); 3) Normal ambient temperature (NAT) and a basal diet; 4) NAT and a basal diet supplemented with 1,000 mg of VC/kg (NAT + VC). Each treatment group contained 10 replicate pens, with 10 broilers per pen. The temperature scheme is shown in Table 1. The VC was purchased from Minsheng Biochemical Technology Co. Ltd. (Hangzhou, Zhejiang province, China), and the content of ascorbic acid was above 98%. The basal diet (mash form) was a corn-soybean meal-based diet and was formulated to meet or exceed nutrient requirements of broilers (Table 2).
Sampling and Measurements Body weight, feed intake, and feed conversion ratio of each replicate were determined on a weekly basis. One replicate (10 birds per replicate) was randomly selected
Table 2. Composition of the basal diet for birds fed 0 to 21 d of age Item Ingredient Corn (%) Soybean meal (%) Soybean oil (%) Fish meal (%) Limestone (%) Calcium hydrophosphate (%) dl-Methionine (%) Mineral premix1 (%) Salt (%) Choline chloride (%) Vitamin premix2 (%) Total (%) Determined composition ME (Mcal/kg) CP (%) Calcium (%) Available phosphorus (%) Methionine (%) Lysine (%) Methionine + cysteine (%)
% 59.2 29.1 4.5 3.8 1 1.24 0.13 0.5 0.35 0.15 0.03 100 3.09 19.7 0.91 0.45 0.45 1.07 0.76
1Provided per kilogram of diet: iron, 100 mg; copper, 8 mg; manganese, 100 mg; zinc, 75 mg; selenium, 0.15 mg; iodine, 0.45 mg. 2Provided per kilogram of diet: vitamin A, 8,000 IU; cholecalciferol, 2,000 IU; vitamin E, 5 IU; vitamin K3, 1 mg; thiamine, 0.4 mg; riboflavin, 3.2 mg; pyridoxine, 1.2 mg; vitamin B12, 6 μg; folic acid, 100 μg; niacin, 7 mg; calcium pantothenate, 5 mg.
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et al. (1993) showed that levels of ascorbic acid (VC) and glutathione in the lungs and liver were low in ascitic broilers housed in a poorly ventilated environment for 7 wk. Wang et al. (2012) recently observed that LAT conditions can induce body hypoxia and result in tissue damage. This study showed an increase in hepatic malondialdehyde (MDA), a biomarker for oxidative stress, and a decrease in the activity of total superoxide dismutase (SOD), an antioxidant that protects against oxidative stress, in ascitic broilers exposed to LAT conditions. Incorporating antioxidants into broilers’ diets has been investigated as an approach to reduce the effects of oxidative stress resulting from hypoxia. Ladmakhi et al. (1997) showed that incorporating VC (500 mg/kg of diet) into the diet of broilers with AS reduced their mortality rates. Another study showed that VC (500 mg/kg of diet) played an important role in decreasing the incidence of AS, which was induced in broilers by LAT conditions and supplemented dietary 3,3c,5-triiodothyronine (Xiang et al., 2002). Considering these previous investigations, the first aim of our study was to determine whether natural LAT conditions were able to induce AS, and how they influenced the blood parameters and antioxidant capacity in broilers. The second aim was to evaluate whether dietary supplemental VC could alleviate the adverse effects of LAT by improving the antioxidant capacity of broilers. This study focused on the starter phase of broilers (1–21 d) to investigate the effects of LAT condition and VC supplementation on the growth performance, blood parameters, and antioxidant capacity in 21-d-old broilers. The results of this study will be important for broiler producers to prevent AS in LAT conditions.
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Statistical Analysis Two-way ANOVA of GLM was used to test the main effects of VC supplement and temperature, and to investigate their interaction effects. A one-way ANOVA
was used to analyze the effect of time on the RBC count, HCT, and HGB level. Duncan’s multiple range test was used as a multiple comparison procedure to compare means when the effect of age was significant. Differences in the means were expressed as either significant (P < 0.05), or trending toward significance (P < 0.1). Statistical analyses were performed using the SAS system (9.0). Data are presented as the mean with pooled SEM values.
RESULTS Effects of Temperature and VC on the Mortality, Heart Index, and Growth Performance of Broilers Broiler mortality under LAT, both with and without the VC supplement, was 8% throughout the study period. Broiler mortality under NAT was 0%. Deaths that occurred between 1 and 14 d were not caused by heart failure or AS, according to the symptoms. After 14 d, one of the broilers in the LAT group (without the VC supplement) died of AS. The morbidities of AS were low in this study according to the apparent symptoms and autopsy, which was only caused by LAT conditions over the entire study period (21 d; data not shown). Heart index values and growth performance measurements are shown in Table 3. The LAT environment resulted in increased heart index values at 21 d (P < 0.05) compared with NAT conditions. There was no effect of VC supplementation on heart index values (P > 0.05). The LAT environment decreased average BW gain during 15 to 21 d (P < 0.01) and increased feed conversion ratio (feed intake/gain, g/g) during the entire period (P < 0.05). The addition of VC to the diet increased ADFI and feed conversion ratio during 1 to 7 d (P < 0.01), but were decreased by the VC supplement during 8 to 14 d. The interactions for ADFI between temperature and VC were significant at 15 to 21 d (P < 0.05), as well as feed conversion ratio at 8 to 14 d (P < 0.01).
Effects of Temperature and VC on the Blood Parameters of Broilers Blood parameter results are shown in Table 4. At 14 d, the LAT environment increased HCT and HGB levels (P < 0.05). Adding the VC supplement increased the RBC count, HCT, and the HGB level at 21 d in the LAT groups and NAT groups (P < 0.05). The interaction for HGB level between temperature and VC tended to be significant at 7 and 21 d (P < 0.1). The blood parameter results varied with broiler age (Table 5). Age of the broiler affected the RBC count, HGB level, and HCT. The RBC count, HGB level, and HCT increased with age in broilers within the LAT + VC group, the NAT group, and the NAT + VC group (P < 0.05).
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from each treatment group at 7, 14, and 21 d for blood collection. Blood (approximately 2 mL) was collected via the vena brachialis and was placed into tubes containing EDTA-Na2 (an anticoagulant) for subsequent determination of blood parameters, which included the hematocrit (HCT), the hemoglobin (HGB) level, and the red blood cell (RBC) count. These blood parameters were tested using an automated hematology analyzer (Abbott CD 3200) within 2 h after collection. Birds were euthanized by cervical dislocation after blood collection. Next, cardiac tissue samples from each treatment were obtained. The atria, major vessels, and gross fat were removed, and the right ventricle was cut away from the left ventricle and septum, which were left intact together. The right ventricle was weighed separately from the left ventricle and septum tissue. Heart index was calculated as the weight of the right ventricle divided by the weight of the left ventricle and septum. On d 21, in addition to the blood samples collected via the vena brachialis, blood samples were also obtained from a jugular vein before euthanasia. The blood samples from the jugular vein were centrifuged (2,000 × g, 10 min, 4°C) to obtain serum. The left liver and lung of each of the selected broilers were obtained. Serum, liver, and lung samples were then stored at −20°C until the antioxidant capacity was analyzed. The antioxidant capacity analyses included measuring VC levels, total antioxidant capacity (T-AOC), MDA levels, amount of protein carbonylation, SOD activity, and glutathione peroxidase (GSH-Px) activity. Serum samples were used directly, whereas the tissue samples were homogenized with saline and centrifuged at 2,000 × g for 10 min at 4°C, and the supernatant was collected for the assay. Spectrophotometry was used to measure the reaction products in the VC and T-AOC assays. The MDA assay was performed using the TBA colorimetric method (Ohkawa et al., 1979). The activity of SOD was measured as the ability of SOD to inhibit the oxidation of oxymine by the xanthine-xanthine oxidase system (Oyanagui, 1984). The activity of GSH-Px was also determined by spectrophotometry to measure the reaction rate of H2O2 and glutathione. The details of the measurement of these components followed the kit instructions (Nanjing Jiancheng Bioengineering Institute, China). In this experiment, broilers that showed abdominal fluid accumulation were regarded as having AS. Broilers that died during the experimental period underwent autopsy to examine whether AS was the cause by observing the tissue lesions and abdominal fluid accumulation. The morbidities and mortalities were recorded.
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Table 3. Effects of temperature and vitamin C (VC) on the heart index, average BW gain (ABWG), ADFI, and feed conversion ratio (feed/gain; FCR) of broilers1 P-value Item
0.19
LAT + VC 0.20
NAT
NAT + VC
0.16
0.16
Temperature
0.01
*
VC
Temperature × VC
0.863
0.457
16.1 32.6 37.9
15.9 30.1 38.9
16.6 36.0 45.9
16.9 35.3 46.4
0.39 2.42 1.68
0.057 0.086 ***
0.900 0.505 0.644
0.566 0.703 0.898
27.9 57.0 101
28.6 50.7 91.5
27.1 54.9 97.4
28.9 40.1 102
0.42 3.67 3.06
0.576 0.092 0.263
** ** 0.452
0.216 0.259 *
0.03 0.05 0.14
** *** **
* *** 0.472
0.777 ** 0.155
1.74 1.77 2.71
1.80 1.70 2.42
1.64 1.53 2.13
1.72 1.14 2.23
1LAT
= low ambient temperature, NAT = normal ambient temperature. *P < 0.05; **P < 0.01; ***P < 0.001.
Effects of Temperature and VC on the Antioxidant Capacity of Broilers Results of antioxidant capacity analyses of the serum, liver, and lungs at 21 d are shown in Table 6. The LAT environment elevated VC level and reduced T-AOC in serum (P < 0.05). The LAT environment tended to increase the levels of MDA and the amount of protein carbonylation in serum (P < 0.1). There were no differences in the activity levels of SOD and GSHPx in serum between the LAT and NAT groups (P > 0.05). Groups that were given the VC supplement tended to have increased T-AOC (P < 0.1), whereas the VC supplement decreased the activity of GSH-Px in serum (P < 0.05). There were no differences in the VC level, MDA level, protein carbonylation level, or the SOD activity in the serum between groups that were given the VC supplement and those that were not (P > 0.05). The LAT environment increased the levels of VC and protein carbonylation, and decreased T-AOC in the liver (P < 0.05). The LAT environment tended to increase the level of MDA in the liver (P < 0.1). There were
no differences in the VC level, MDA level, T-AOC, or amount of protein carbonylation in the liver between groups that were given the VC supplement and those that were not (P > 0.05). In the lungs, LAT environment increased the MDA level and the amount of protein carbonylation, and decreased T-AOC (P < 0.05). There were no differences in the VC level in the lungs between the LAT and NAT groups (P > 0.05), and no effects of the VC supplementation were observed on the VC level, MDA level, T-AOC, and amount of protein carbonylation in the lung (P > 0.05).
DISCUSSION Considering that LAT conditions are a major stressor for broilers in the winter and contribute to the high occurrence of AS in most areas, it was worthwhile to investigate the response of broilers to natural LAT conditions. The heart index value was indicative of AS because right ventricular hypertrophy is an indicator of pulmonary hypertension associated with AS (Huchzer-
Table 4. Effects of temperature and vitamin C (VC) on the blood parameters of broilers1 P-value Item HGB (g/L) 7d 14 d 21 d HCT (%) 7d 14 d 21 d RBC (× 1012/L) 7d 14 d 21 d 1LAT
LAT
LAT + VC
NAT
NAT + VC
Pooled SEM
77.6 94.7 86.7
72.2 91.0 104
71.0 79.7 90.8
75.8 81.3 96.8
2.42 4.44 2.46
0.543 * 0.613
0.903 0.828 **
0.051 0.565 0.058
Temperature
VC
Temperature × VC
0.32 0.36 0.34
0.31 0.35 0.40
0.30 0.31 0.36
0.31 0.31 0.38
0.01 0.02 0.01
0.146 * 0.946
0.908 0.854 *
0.215 0.585 0.157
2.28 2.64 2.57
2.16 2.57 2.92
2.10 2.32 2.66
2.24 2.40 2.79
0.08 0.12 0.07
0.544 0.068 0.753
0.886 0.967 **
0.108 0.519 0.127
= low ambient temperature; NAT = normal ambient temperature; HGB = hemoglobin; HCT = hematocrit; RBC = red blood cell. *P < 0.05. **P < 0.01.
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Heart index (g/g) 21 d ABWG (g) 1–7 d 8–14 d 15–21 d ADFI (g) 1–7 d 8–14 d 15–21 d FCR (g/g) 1–7 d 8–14 d 15–21 d
LAT
Pooled SEM
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YANG ET AL. Table 5. Effect of broiler age on red blood cell (RBC) count, hemoglobin (HGB) level, and hematocrit (HCT)1 Item
LAT 1012/L)
2.28b 2.64a 2.57ab 0.10 *
2.16c 2.57b 2.92a 0.10 ***
77.6b 94.7a 86.7ab 3.19 **
NAT + VC
2.10c 2.32b 2.66a 0.04 ***
72.2c 91.0b 104a 3.24 ***
0.32b 0.36a 0.34ab 0.01 *
NAT
2.24b 2.40b 2.79a 0.10 *
71.0c 79.7b 90.8a 1.70 ***
0.31b 0.35ab 0.40a 0.02 **
75.8b 81.3b 96.8a 3.70 **
0.30b 0.31b 0.36a 0.01 **
0.31b 0.31b 0.38a 0.01 **
a–cDifferent
lowercase letters in the same column mean significant differences (P < 0.05). = low ambient temperature; NAT = normal ambient temperature; VC = vitamin C. *P < 0.05. **P < 0.01. ***P < 0.001. 1LAT
meyer and De Ruyck, 1986). A previous study showed that the effect of LAT on the right ventricle was remarkable, resulting in heavier total ventricular weights and higher values for pulmonary arterial pressure (Wideman and Tackett, 2000). The heart index was generally increased with age or BW. Because LAT stress resulted in poor growth in the present experiment, the heart index values of both LAT and NAT treatments were low at 21 d. However, LAT increased heart index values compared with NAT. Therefore, these results implied that LAT caused heavier right ventricle, and it increased the possibility of developing AS.
Hypoxia has been shown to result in significant reductions in BW, which are caused by increased energy demands (Yahav and McMurtry, 2001). In the current study, hypoxia resulting from LAT conditions caused an increase in the broilers’ feed conversion ratio because the birds needed more energy to produce heat to maintain their temperature and depressed their BW gain, implying a strong LAT stress on broilers according to the growth performance. The addition of VC decreased the feed conversion ratio by reducing the ADFI during 8 to 14 d. However, there were no differences in growth performance after 14 d as a result of adding VC.
Table 6. Effects of temperature and vitamin C (VC) on the VC, total antioxidant capacity (T-AOC), malondialdehyde (MDA), protein carbonylation, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in the serum and on the VC, T-AOC, MDA, and protein carbonylation in the liver and the lungs at 21 d1 P-value
Item Serum VC (μg/mL) T-AOC (U/mL) SOD (U/mL) GSH-Px (U/mL) MDA (nmol/mL) Protein carbonylation (nmol/mL) Liver VC (μg/mg of protein) T-AOC (U/mg of protein) MDA (nmol/mg of protein) Protein carbonylation (nmol/mg of protein) Lungs VC (μg/mg of protein) T-AOC (U/mg of protein) MDA (nmol/mg of protein) Protein carbonylation (nmol/mg of protein) 1LAT
LAT
LAT + VC
NAT
NAT + VC
Pooled SEM
Temperature
VC
Temperature × VC
16.0 3.01 130 194 15.8 2.03
18.2 4.62 141 154 15.6 1.95
13.2 4.78 151 193 14.2 1.87
14.8 4.89 151 159 10.4 1.20
1.42 0.46 17.2 16.4 1.74 0.23
* * 0.372 0.909 0.070 0.063
0.207 0.082 0.766 * 0.275 0.124
0.839 0.127 0.741 0.841 0.317 0.217
2.09 0.82 1.04 4.11
2.21 0.94 0.82 3.98
1.58 0.98 0.47 2.56
1.80 1.07 0.73 3.09
0.18 0.06 0.17 0.45
* * 0.067 *
0.346 0.111 0.926 0.666
0.780 0.841 0.176 0.476
0.06 0.28 3.13 10.7
0.09 0.40 2.32 11.8
0.21 0.60 1.47 6.82
0.08 0.64 1.70 7.19
0.05 0.10 0.37 1.96
0.148 * ** *
0.339 0.434 0.439 0.708
0.098 0.670 0.174 0.847
= low ambient temperature; NAT = normal ambient temperature. *P < 0.05. **P < 0.01.
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RBC (× 7d 14 d 21 d Pooled SEM P-value HGB (g/L) 7d 14 d 21 d Pooled SEM P-value HCT (%) 7d 14 d 21 d Pooled SEM P-value
LAT + VC
EFFECTS OF LOW TEMPERATURES AND VITAMIN C ON BROILERS
Hypoxia that is induced by LAT conditions can also result in oxidation reactions. When oxidation overcomes the body’s ability to protect itself using antioxidants, oxidative stress occurs (Iqbal et al., 2002). Mujahid and Furuse (2009) have observed some oxidative stress in the brains and hearts of broilers exposed to LAT conditions. The MDA compound is an end product of the oxidative degradation of lipids and is an indicator of oxidative stress (Del Rio et al., 2005). Heat stress, which causes hypoxia, has been shown to increase MDA levels in liver tissue, both in vitro and in vivo (Feng et al., 2008). One study found that when broilers were exposed to LAT environment for 3 wk, MDA level in the plasma increased (Pan et al., 2005). Along with the MDA level, the amount of protein carbonylation in the body, which is a form of protein oxidation, is also an indicator of the body’s degree of oxidation. In agreement with Aruoma (1994), it shown that free radicals can oxidize both lipids and proteins, worsening disease conditions in broilers. In our study, LAT conditions also decreased T-AOC in the serum, liver, and lungs, but significantly increased the VC level in serum and liver. It is possible that the hypoxic conditions resulted in an inflammatory response, as the level of VC is usually higher in regions of inflammation than in areas that are not the focus of an inflammation response. In tissues and cells, many specific enzymes, such as SOD and GSH-Px, can eliminate ROS that arise from oxidative stress. The activity of GSH-Px in the mitochondria of the lungs and in the liver tissue of broilers with AS have been found to be higher than its activity in normal broilers (Iqbal et al., 2002), indicating that the amount of GSH-Px in tissues increases to eliminate ROS when necessary. However, when the amount of ROS in mitochondria exceeded the antioxidant abilities provided by SOD and GSH-Px activities, mitochondrial injury would occur (Bottje and Wideman, 1995). However, Wang et al. (2012) reported that the total SOD activity in the liver significantly decreased in broilers with AS, which indicates that AS may severely damage the mechanism for SOD production in the liver. In our experiment, the activities of SOD and GSH-Px in the serum were similar under both LAT and NAT conditions. This result might be due to the short study period (21 d) and that fact that only LAT was used as an AS-inducing factor. Vitamin C has been found to be a good scavenger of oxygen (Cort, 1974), and it can alleviate oxidative stress. However, one study showed that VC (100 mg/ kg of diet) had no significant effect on the level of SOD in the serum (Tras et al., 2000). In our study, the activity of GSH-Px in serum decreased upon addition of VC to the diet. Our results are in accordance with results from a previous study, where dietary vitamin E reduced the activity of GSH-Px in ascitic broilers as this vitamin acts also as an antioxidant in vivo (Iqbal et al., 2002). The antioxidant effects of VC were only evident as indicated by the increasing T-AOC levels in the current study. Because oxidative stress caused by
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A similar result was reported in a previous study, which revealed that adding VC (500 mg/kg) to the diet had no significant effect on growth performance (Ladmakhi et al., 1997). Shlosberg et al. (1992) reported that LAT stress caused elevated HCT, which would lead to AS. Wang et al. (2012) reported that HCT, HGB levels, and RBC counts of broilers with AS were higher than those of healthy broilers, suggesting that hypoxic conditions caused the levels of these substances to increase to fulfill tissues’ demand for oxygen. Increases in the HCT and HGB levels have been observed in broilers in LAT conditions, and blood volume values were significantly elevated at 10°C than at 20°C (Yahav et al., 1997; Wideman et al., 1998; Wideman and Tackett, 2000; Luger et al., 2001). In the present study, LAT conditions markedly increased the HCT and HGB levels at 14 d, suggesting that LAT conditions had elevated the possibility of birds developing AS. Even though many studies have investigated the levels of these blood parameters related to AS, there are few reports about the effect of VC on blood parameters of broilers. In calves experiencing heat stress, one study showed that a VC supplementation (20 g/d) decreased their RBC counts, HCT, and HGB levels (Kim et al., 2012). When investigating these blood parameters in broilers, Tras et al. (2000) found that VC (100 mg/ kg of diet) had no significant effect. In our study, RBC counts, HCT, and HGB levels in broilers that were given a VC supplement increased at 21 d, especially for broilers in LAT conditions. This was a compensation mechanism. Because in some condition, the lower HCT level indicated less erythrocytes in blood volume, and it induced a greater risk of hypoxia. Broilers with lower HCT level had significantly higher mortality other than mortality caused by AS (Shlosberg and Bellaiche, 1996). The increase of RBC counts, HGB, and HCT levels due to VC addition were not anticipated; therefore, the mechanism was not investigated in this study. These results provide further evidence that the increase in RBC counts, HCT, and HGB levels induced by LAT conditions resulted from the increased need for oxygen in broilers. Hypoxia has been shown to increase the amount of reactive oxygen species (ROS) in cells and mitochondria and disturb the oxidation-reduction system in the body, as well as stabilize the hypoxia-inducible factor, leading to vascular remodeling, which is the foundation of AS (Chandel et al., 1998, 2000). Free radicals or ROS can result in lipid and protein peroxidation, and if it is not reduced by antioxidant protective mechanisms, tissues would be damaged. Supplementation with VC has been found to alleviate the oxidative damage induced by ROS and therefore allow cells to maintain their proper redox state (Blagojeviü, 2007). Broilers living in conditions of low ventilation have been reported as having low levels of VC in their liver and lungs, which suggests that VC is consumed in the oxidation reactions caused by hypoxia (Enkvetchakul et al., 1993).
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it needs further study when broilers are reared under LAT conditions.
ACKNOWLEDGMENTS We thank Todd. J. Applegate and Chen Xi (Animal Science Department, Purdue University, West Lafayette, IN) for their role in revising the paper. This research was supported by grants from the National Natural Science Foundation of China (31101733).
REFERENCES Aengwanich, W., and S. Simaraks. 2004. Pathology of heart, lung, liver and kidney in broilers under chronic heat stress. Pathology 26:418. Aruoma, O. I. 1994. Free radicals and antioxidant strategies in sports. J. Nutr. Biochem. 5:370–381. Bautista-Ortega, J., and C. A. Ruiz-Feria. 2010. l-Arginine and antioxidant vitamins E and C improve the cardiovascular performance of broiler chickens grown under chronic hypobaric hypoxia. Poult. Sci. 89:2141–2146. Blagojeviü, D. P. 2007. Antioxidant systems in supporting environmental and programmed adaptations to low temperatures. Cryo Letters 28:137–150. Bottje, W. G., and R. F. Wideman. 1995. Potential role of free radicals in the pathogenesis of pulmonary hypertension syndrome. Poult. Avian Biol. Rev. 6:211–231. Carr, A. C., B. Z. Zhu, and B. Frei. 2000. Potential antiatherogenic mechanisms of ascorbate (vitamin C) and α-tocopherol (vitamin E). Circ. Res. 87:349–354. Chandel, N. S., E. Maltepe, E. Goldwasser, C. E. Mathieu, M. C. Simon, and P. T. Schumacker. 1998. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc. Natl. Acad. Sci. USA 95:11715–11720. Chandel, N. S., D. S. McClintock, C. E. Feliciano, T. M. Wood, J. A. Melendez, A. M. Rodriguez, and P. T. Schumacker. 2000. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1α during hypoxia. J. Biol. Chem. 275:25130–25138. Cort, W. M. 1974. Antioxidant activity of tocopherols, ascorbyl palmitate, and ascorbic acid and their mode of action. J. Am. Oil Chem. Soc. 51:321–325. Daneshyar, M., H. Kermanshahi, and A. Golian. 2009. Changes of biochemical parameters and enzyme activities in broiler chickens with cold-induced ascites. Poult. Sci. 88:106–110. Decuypere, E., C. Vega, T. Bartha, J. Buyse, J. Zoons, and G. A. A. Albers. 1994. Increased sensitivity to triiodothyronine (T3) of broiler lines with a high susceptibility for ascites. Br. Poult. Sci. 35:287–297. Del Rio, D., A. J. Stewart, and N. Pellegrini. 2005. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr. Metab. Cardiovasc. Dis. 15:316–328. Enkvetchakul, B., W. Bottje, N. Anthony, R. Moore, and W. Huff. 1993. Compromised antioxidant status associated with ascites in broilers. Poult. Sci. 72:2272–2280. Feng, J., M. Zhang, S. Zheng, P. Xie, and A. Ma. 2008. Effects of high temperature on multiple parameters of broilers in vitro and in vivo. Poult. Sci. 87:2133–2139. Guney, M., B. Oral, H. Demirin, N. Karahan, T. Mungan, and N. Delibas. 2007. Protective effects of vitamins C and E against endometrial damage and oxidative stress in fluoride intoxication. Clin. Exp. Pharmacol. Physiol. 34:467–474. Huchzermeyer, F. W., and A. M. De Ruyck. 1986. Pulmonary hypertension syndrome associated with ascites in broilers. Vet. Rec. 119:94. Iqbal, M., D. Cawthon, K. Beers, R. F. Wideman, and W. G. Bottje. 2002. Antioxidant enzyme activities and mitochondrial fatty
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LAT conditions was severe, supplemental VC did not improve the antioxidant capacity. Vitamin C generally acted as a co-antioxidant that functioned by cooperating with other antioxidants, for instance, with vitamin E. Vitamin C was shown to restore the antioxidant capability of vitamin E by converting the tocopherol radical back to its reduced state (Witting and Horwitt, 1964; Carr et al., 2000; Guney et al., 2007). Vitamin C cooperating with vitamin E can become a scavenger of ROS to protect NO, which was a substance maintaining pulmonary vasodilatation (Ruiz-Feria, 2009; Bautista-Ortega and Ruiz-Feria, 2010). In our study, vitamin E was not added to the basal diet, and soybean oil was included, which was apt to be oxidized. Vitamin C with inappropriate supplementation can also become a pro-oxidative agent by reducing Fe3+ to Fe2+, which then generated free radicals by interacting with Cu2+ (Bottje and Wideman, 1995). This suggests that the proper cooperative substance and the additive amount of VC should be continually explored when the broilers are reared under LAT conditions. The change in antioxidant capacity (T-AOC) observed in this study indicated that hypoxia, induced by LAT conditions, had a marked effect on the different tissues, especially the lungs. Edema and hyperemia of the lungs have been observed in broilers experiencing chronic heat stress, which causes hypoxia, and hemorrhaging was observed in their alveoli, indicating that lung lesions are common consequences of hypoxia (Aengwanich and Simaraks, 2004). When AS is present, cell growth and migration of pulmonary arterioles have been found (Nain et al., 2009); pulmonary arterioles become more muscular, evident through observation of thicker media and cellular proliferation in the intima, which results in pulmonary hypertension (Xiang et al., 2002). In addition, a study by Iqbal et al. (2001) showed that hydrogen peroxide, another biomarker of oxidative stress, was overproduced in the lung mitochondria of broilers experiencing pulmonary hypertension syndrome (Iqbal et al., 2001). Similarly, the present study showed that LAT conditions worsened the oxidative stress in broilers, as evidenced by changes in the levels of T-AOC, MDA, and protein carbonylation. Although it has been reported that VC (500 mg/kg of diet) can alleviate AS by reducing the muscularization of the pulmonary arterioles (Xiang et al., 2002), it had little effect on maintaining proper lung function in the present study. In conclusion, the natural LAT scheme employed in this experiment caused oxidative stress of broilers over a 21-d period, as shown by the significant increase in HCT, poor growth, and low antioxidant capacities. Although the LAT conditions failed to induce large-scale incidence of AS, it can be used as a model for future studies that focusing on the mechanisms by which hypoxia leads to oxidative stress. The effect of the dietary VC supplementation on the antioxidant capacity of 21-d-old broilers was not significant, indicating that
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Oyanagui, Y. 1984. Reevaluation of assay methods and establishment of kit for superoxide dismutase activity. Anal. Biochem. 142:290–296. Pan, J. Q., X. Tan, J. C. Li, W. D. Sun, and X. L. Wang. 2005. Effects of early feed restriction and cold temperature on lipid peroxidation, pulmonary vascular remodelling and ascites morbidity in broilers under normal and cold temperature. Br. Poult. Sci. 46:374–381. Ruiz-Feria, C. A. 2009. Concurrent supplementation of arginine, vitaminE, and vitamin C improve cardiopulmonary performance in broilers chickens. Poult. Sci. 88:526–535. Shlosberg, A., and M. Bellaiche. 1996. Hematocrit values and mortality from ascites in cold-stressed broilers from parents selected by hematocrit. Poult. Sci. 75:1–5. Shlosberg, A., I. Zadikov, U. Bendheim, V. Handji, and E. Berman. 1992. The effects of poor ventilation, low temperatures, type of feed and sex of bird on the development of ascites in broilers. Physiopathological factors. Avian Pathol. 21:369–382. Tras, B., F. Inal, A. L. Bas, V. Altunok, M. Elmas, and E. Yazar. 2000. Effects of continuous supplementations of ascorbic acid, aspirin, vitamin E and selenium on some haematological parameters and serum superoxide dismutase level in broiler chickens. Br. Poult. Sci. 41:664–666. Wang, Y., Y. Guo, D. Ning, Y. Peng, H. Cai, J. Tan, Y. Yang, and D. Liu. 2012. Changes of hepatic biochemical parameters and proteomics in broilers with cold-induced ascites. J. Anim. Sci. Biotechnol. 3:41. Wideman, R. F. Jr., and C. D. Tackett. 2000. Cardio-pulmonary function in broilers reared at warm or cool temperatures: Effect of acute inhalation of 100% oxygen. Poult. Sci. 79:257–264. Wideman, R. F. Jr., T. Wing, Y. K. Kirby, M. F. Forman, N. Marson, C. D. Tackett, and C. A. Ruiz-Feria. 1998. Evaluation of minimally invasive indices for predicting ascites susceptibility in three successive hatches of broilers exposed to cool temperatures. Poult. Sci. 77:1565–1573. Witting, L. A., and M. K. Horwitt. 1964. Effect of degree of fatty acid unsaturation in tocopherol deficiency-induced creatinuria. J. Nutr. 82:19. Xiang, R. P., W. D. Sun, J. Y. Wang, and X. L. Wang. 2002. Effect of vitamin C on pulmonary hypertension and muscularisation of pulmonary arterioles in broilers. Br. Poult. Sci. 43:705–712. Yahav, S., and J. P. McMurtry. 2001. Thermotolerance acquisition in broiler chickens by temperature conditioning early in life–the effect of timing and ambient temperature. Poult. Sci. 80:1662– 1666. Yahav, S., A. Straschnow, I. Plavnik, and S. Hurwitz. 1997. Blood system response of chickens to changes in environmental temperature. Poult. Sci. 76:627–633.
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acids in pulmonary hypertension syndrome (PHS) in broilers. Poult. Sci. 81:252–260. Iqbal, M., D. Cawthon, R. F. Wideman, and W. G. Bottje. 2001. Lung mitochondrial dysfunction in pulmonary hypertension syndrome. I. Site-specific defects in the electron transport chain. Poult. Sci. 80:485–495. Julian, R. J., G. W. Friars, H. French, and M. Quinton. 1987. The relationship of right ventricular hypertrophy, right ventricular failure, and ascites to weight gain in broiler and roaster chickens. Avian Dis. 31:130–135. Julian, R. J., I. McMillan, and M. Quinton. 1989. The effect of cold and dietary energy on right ventricular hypertrophy, right ventricular failure and ascites in meat-type chickens. Avian Pathol. 18:675–684. Julian, R. J., and E. J. Squires. 1994. Haematopoietic and right ventricular response to intermittent hypobaric hypoxia in meat-type chickens. Avian Pathol. 23:539–545. Kim, J. H., L. L. Mamuad, C. J. Yang, S. H. Kim, J. K. Ha, W. S. Lee, K. K. Cho, and S. S. Lee. 2012. Hemato-biochemical and cortisol profile of Holstein growing-calves supplemented with vitamin C during summer season. J. Anim. Sci. 25:361–368. Ladmakhi, M. H., N. Buys, E. Dewil, G. Rahimi, and E. Decuypere. 1997. The prophylactic effect of vitamin C supplementation on broiler ascites incidence and plasma thyroid hormone concentration. Avian Pathol. 26:33–44. Luger, D., D. Shinder, V. Rzepakovsky, M. Rusal, and S. Yahav. 2001. Association between weight gain, blood parameters, and thyroid hormones and the development of ascites syndrome in broiler chickens. Poult. Sci. 80:965–971. Maxwell, M. H., and G. W. Robertson. 1997. World broiler ascites survey 1996. Poult. Int. 36:16–33. Mujahid, A., and M. Furuse. 2009. Oxidative damage in different tissues of neonatal chicks exposed to low environmental temperature. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 152:604– 608. Nain, S., B. Ling, B. Bandy, J. Alcorn, C. Wojnarowicz, B. Laarveld, and A. A. Olkowski. 2008. The role of oxidative stress in the development of congestive heart failure in a chicken genotype selected for rapid growth. Avian Pathol. 37:367–373. Nain, S., C. Wojnarowicz, B. Laarveld, and A. A. Olkowski. 2009. Vascular remodeling and its role in the pathogenesis of ascites in fast growing commercial broilers. Res. Vet. Sci. 86:479–484. Ohkawa, H., N. Ohishi, and K. Yagi. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95:351–358. Owen, R. L., R. F. Wideman Jr., A. L. Hattel, and B. S. Cowen. 1990. Use of a hypobaric chamber as a model system for investigating ascites in broilers. Avian Dis. 34:754–758.
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Key words: low ambient temperature, vitamin C, ascites, blood parameter, antioxidant capacity 2014 Poultry Science 93:898–905 http://dx.doi.org/10.3382/ps.2013-03438 temperature by 1°C per day from 5 to 22 d resulted in broilers experiencing right ventricular hypertrophy and death from AS. In another study, keeping ambient temperatures at 32 and 30°C during 1 and 2 wk, respectively, and then reducing it to 15°C during the third week, also led to more AS incidences in broilers (Daneshyar et al., 2009). Because of the significant temperature changes in the winter in China, the expensive thermal equipment to mitigate temperature effects, and the high incidence of AS affecting broilers in winter, it is important to study the effects of natural temperatures on the incidence of AS in winter. Previous studies have indicated that a lack of oxygen in the body of modern broilers, due the imbalance between rapid growth and limited cardiopulmonary development, is a major factor in developing AS (Julian et al., 1987; Nain et al., 2009). Nain et al. (2008) reported that fast-growing commercial broilers had a high risk of heart failure coupled with increased oxidative stress resulting from continuous hypoxia; oxidative stress caused damage to tissues and exhausted the broilers’ antioxidant supply. Previously, Enkvetchakul
INTRODUCTION Fast-growing broilers are susceptible to ascites syndrome (AS), which causes fluid accumulation in their abdominal cavities and is associated with pathological damage to the heart, liver, and lungs. This syndrome leads to increased mortality and results in substantial economic losses (Maxwell and Robertson, 1997). Hypobaric chambers (Owen et al., 1990; Julian and Squires, 1994), low ventilation (Enkvetchakul et al., 1993), and the compound 3,3c,5-triiodothyronine (Decuypere et al., 1994) have all been found to induce AS. Low ambient temperatures (LAT), which affect most farming areas in the winter, have also been reported to induce widespread incidences of AS. In a study by Julian et al. (1989), holding meat-type chickens at an ambient temperature of 30°C and then reducing the
©2014 Poultry Science Association Inc. Received June 22, 2013. Accepted December 16, 2013. 1 Corresponding author: [email protected]
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whole experimental period (P < 0.01), whereas it increased heart index at 21 d (P < 0.05) and hematocrit and hemoglobin level at 14 d (P < 0.05). Supplementing the diet with VC increased hematocrit, hemoglobin, and red blood cell count at 21 d (P < 0.05). At 21 d, LAT conditions decreased total antioxidant capacity in the serum, liver, and lungs (P < 0.05), and it also increased the levels of VC in the serum and liver, the amount of protein carbonylation in liver and lungs, and the malondialdehyde level in the lungs (P < 0.05). The addition of VC tended to increase the total antioxidant capacity level in serum (P < 0.1). Low ambient temperature resulted in oxidative stress for broilers that were fed from 1 to 21 d of age, whereas no significant effect was found on the antioxidant activity by dietary VC supplementation.
ABSTRACT The study was conducted to determine the effects of low ambient temperature (LAT) and a vitamin C (VC) dietary supplement on the growth performance, blood parameters, and antioxidant capacity of 21-d-old broilers. A total of 400 one-day-old male Cobb broilers were assigned to 1 of 4 treatments as follows: 1) LAT and a basal diet; 2) LAT and a basal diet supplemented with 1,000 mg of VC/kg (LAT + VC); 3) normal ambient temperature (NAT) and a basal diet; 4) NAT and a basal diet supplemented with 1,000 mg of VC/kg (NAT + VC). All birds were fed to 21 d of age. Broilers in groups 1 and 2 were raised at 24 to 26°C during 1 to 7 d, and at 9 to 11°C during 8 to 21 d, whereas groups 3 and 4 were raised at 29 to 31°C during 1 to 7 d and at 24 to 26°C during 8 to 21 d. The LAT increased the feed conversion ratio during the
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EFFECTS OF LOW TEMPERATURES AND VITAMIN C ON BROILERS Table 1. Temperature scheme1 Indoor temperature
Temperature inside the cage
Item
0–7 d
8–21 d
0–7 d
8–21 d
LAT (°C) NAT (°C)
24–26 29–31
9–11 24–26
27–29 31–33
11–13 27–29
1LAT
= low ambient temperature, NAT = normal ambient temperature.
MATERIALS AND METHODS Birds, Experimental Design, and Diet The animal experiment was carried out in accordance with the Chinese guidelines for animal welfare and approved by the Animal Health and Care Committee of Sichuan Agricultural University. A total of 400 one-day-old male Cobb broilers were purchased from a commercial hatchery (Wenjiang Zhengda Co. Ltd., Chengdu, Sichuan province, China). They were raised in cage pens (1.5 m × 1 m × 0.5 m) at the farm of the Animal Nutrition Institute of Sichuan Agricultural
University in China. The initial BW was similar among all pens. Feed and water were provided ad libitum. The experimental treatments were arranged as a 2 × 2 factorial as follows: 1) LAT and a basal diet; 2) LAT and a basal diet supplemented with 1,000 mg of VC/kg (LAT + VC); 3) Normal ambient temperature (NAT) and a basal diet; 4) NAT and a basal diet supplemented with 1,000 mg of VC/kg (NAT + VC). Each treatment group contained 10 replicate pens, with 10 broilers per pen. The temperature scheme is shown in Table 1. The VC was purchased from Minsheng Biochemical Technology Co. Ltd. (Hangzhou, Zhejiang province, China), and the content of ascorbic acid was above 98%. The basal diet (mash form) was a corn-soybean meal-based diet and was formulated to meet or exceed nutrient requirements of broilers (Table 2).
Sampling and Measurements Body weight, feed intake, and feed conversion ratio of each replicate were determined on a weekly basis. One replicate (10 birds per replicate) was randomly selected
Table 2. Composition of the basal diet for birds fed 0 to 21 d of age Item Ingredient Corn (%) Soybean meal (%) Soybean oil (%) Fish meal (%) Limestone (%) Calcium hydrophosphate (%) dl-Methionine (%) Mineral premix1 (%) Salt (%) Choline chloride (%) Vitamin premix2 (%) Total (%) Determined composition ME (Mcal/kg) CP (%) Calcium (%) Available phosphorus (%) Methionine (%) Lysine (%) Methionine + cysteine (%)
% 59.2 29.1 4.5 3.8 1 1.24 0.13 0.5 0.35 0.15 0.03 100 3.09 19.7 0.91 0.45 0.45 1.07 0.76
1Provided per kilogram of diet: iron, 100 mg; copper, 8 mg; manganese, 100 mg; zinc, 75 mg; selenium, 0.15 mg; iodine, 0.45 mg. 2Provided per kilogram of diet: vitamin A, 8,000 IU; cholecalciferol, 2,000 IU; vitamin E, 5 IU; vitamin K3, 1 mg; thiamine, 0.4 mg; riboflavin, 3.2 mg; pyridoxine, 1.2 mg; vitamin B12, 6 μg; folic acid, 100 μg; niacin, 7 mg; calcium pantothenate, 5 mg.
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et al. (1993) showed that levels of ascorbic acid (VC) and glutathione in the lungs and liver were low in ascitic broilers housed in a poorly ventilated environment for 7 wk. Wang et al. (2012) recently observed that LAT conditions can induce body hypoxia and result in tissue damage. This study showed an increase in hepatic malondialdehyde (MDA), a biomarker for oxidative stress, and a decrease in the activity of total superoxide dismutase (SOD), an antioxidant that protects against oxidative stress, in ascitic broilers exposed to LAT conditions. Incorporating antioxidants into broilers’ diets has been investigated as an approach to reduce the effects of oxidative stress resulting from hypoxia. Ladmakhi et al. (1997) showed that incorporating VC (500 mg/kg of diet) into the diet of broilers with AS reduced their mortality rates. Another study showed that VC (500 mg/kg of diet) played an important role in decreasing the incidence of AS, which was induced in broilers by LAT conditions and supplemented dietary 3,3c,5-triiodothyronine (Xiang et al., 2002). Considering these previous investigations, the first aim of our study was to determine whether natural LAT conditions were able to induce AS, and how they influenced the blood parameters and antioxidant capacity in broilers. The second aim was to evaluate whether dietary supplemental VC could alleviate the adverse effects of LAT by improving the antioxidant capacity of broilers. This study focused on the starter phase of broilers (1–21 d) to investigate the effects of LAT condition and VC supplementation on the growth performance, blood parameters, and antioxidant capacity in 21-d-old broilers. The results of this study will be important for broiler producers to prevent AS in LAT conditions.
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Statistical Analysis Two-way ANOVA of GLM was used to test the main effects of VC supplement and temperature, and to investigate their interaction effects. A one-way ANOVA
was used to analyze the effect of time on the RBC count, HCT, and HGB level. Duncan’s multiple range test was used as a multiple comparison procedure to compare means when the effect of age was significant. Differences in the means were expressed as either significant (P < 0.05), or trending toward significance (P < 0.1). Statistical analyses were performed using the SAS system (9.0). Data are presented as the mean with pooled SEM values.
RESULTS Effects of Temperature and VC on the Mortality, Heart Index, and Growth Performance of Broilers Broiler mortality under LAT, both with and without the VC supplement, was 8% throughout the study period. Broiler mortality under NAT was 0%. Deaths that occurred between 1 and 14 d were not caused by heart failure or AS, according to the symptoms. After 14 d, one of the broilers in the LAT group (without the VC supplement) died of AS. The morbidities of AS were low in this study according to the apparent symptoms and autopsy, which was only caused by LAT conditions over the entire study period (21 d; data not shown). Heart index values and growth performance measurements are shown in Table 3. The LAT environment resulted in increased heart index values at 21 d (P < 0.05) compared with NAT conditions. There was no effect of VC supplementation on heart index values (P > 0.05). The LAT environment decreased average BW gain during 15 to 21 d (P < 0.01) and increased feed conversion ratio (feed intake/gain, g/g) during the entire period (P < 0.05). The addition of VC to the diet increased ADFI and feed conversion ratio during 1 to 7 d (P < 0.01), but were decreased by the VC supplement during 8 to 14 d. The interactions for ADFI between temperature and VC were significant at 15 to 21 d (P < 0.05), as well as feed conversion ratio at 8 to 14 d (P < 0.01).
Effects of Temperature and VC on the Blood Parameters of Broilers Blood parameter results are shown in Table 4. At 14 d, the LAT environment increased HCT and HGB levels (P < 0.05). Adding the VC supplement increased the RBC count, HCT, and the HGB level at 21 d in the LAT groups and NAT groups (P < 0.05). The interaction for HGB level between temperature and VC tended to be significant at 7 and 21 d (P < 0.1). The blood parameter results varied with broiler age (Table 5). Age of the broiler affected the RBC count, HGB level, and HCT. The RBC count, HGB level, and HCT increased with age in broilers within the LAT + VC group, the NAT group, and the NAT + VC group (P < 0.05).
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from each treatment group at 7, 14, and 21 d for blood collection. Blood (approximately 2 mL) was collected via the vena brachialis and was placed into tubes containing EDTA-Na2 (an anticoagulant) for subsequent determination of blood parameters, which included the hematocrit (HCT), the hemoglobin (HGB) level, and the red blood cell (RBC) count. These blood parameters were tested using an automated hematology analyzer (Abbott CD 3200) within 2 h after collection. Birds were euthanized by cervical dislocation after blood collection. Next, cardiac tissue samples from each treatment were obtained. The atria, major vessels, and gross fat were removed, and the right ventricle was cut away from the left ventricle and septum, which were left intact together. The right ventricle was weighed separately from the left ventricle and septum tissue. Heart index was calculated as the weight of the right ventricle divided by the weight of the left ventricle and septum. On d 21, in addition to the blood samples collected via the vena brachialis, blood samples were also obtained from a jugular vein before euthanasia. The blood samples from the jugular vein were centrifuged (2,000 × g, 10 min, 4°C) to obtain serum. The left liver and lung of each of the selected broilers were obtained. Serum, liver, and lung samples were then stored at −20°C until the antioxidant capacity was analyzed. The antioxidant capacity analyses included measuring VC levels, total antioxidant capacity (T-AOC), MDA levels, amount of protein carbonylation, SOD activity, and glutathione peroxidase (GSH-Px) activity. Serum samples were used directly, whereas the tissue samples were homogenized with saline and centrifuged at 2,000 × g for 10 min at 4°C, and the supernatant was collected for the assay. Spectrophotometry was used to measure the reaction products in the VC and T-AOC assays. The MDA assay was performed using the TBA colorimetric method (Ohkawa et al., 1979). The activity of SOD was measured as the ability of SOD to inhibit the oxidation of oxymine by the xanthine-xanthine oxidase system (Oyanagui, 1984). The activity of GSH-Px was also determined by spectrophotometry to measure the reaction rate of H2O2 and glutathione. The details of the measurement of these components followed the kit instructions (Nanjing Jiancheng Bioengineering Institute, China). In this experiment, broilers that showed abdominal fluid accumulation were regarded as having AS. Broilers that died during the experimental period underwent autopsy to examine whether AS was the cause by observing the tissue lesions and abdominal fluid accumulation. The morbidities and mortalities were recorded.
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Table 3. Effects of temperature and vitamin C (VC) on the heart index, average BW gain (ABWG), ADFI, and feed conversion ratio (feed/gain; FCR) of broilers1 P-value Item
0.19
LAT + VC 0.20
NAT
NAT + VC
0.16
0.16
Temperature
0.01
*
VC
Temperature × VC
0.863
0.457
16.1 32.6 37.9
15.9 30.1 38.9
16.6 36.0 45.9
16.9 35.3 46.4
0.39 2.42 1.68
0.057 0.086 ***
0.900 0.505 0.644
0.566 0.703 0.898
27.9 57.0 101
28.6 50.7 91.5
27.1 54.9 97.4
28.9 40.1 102
0.42 3.67 3.06
0.576 0.092 0.263
** ** 0.452
0.216 0.259 *
0.03 0.05 0.14
** *** **
* *** 0.472
0.777 ** 0.155
1.74 1.77 2.71
1.80 1.70 2.42
1.64 1.53 2.13
1.72 1.14 2.23
1LAT
= low ambient temperature, NAT = normal ambient temperature. *P < 0.05; **P < 0.01; ***P < 0.001.
Effects of Temperature and VC on the Antioxidant Capacity of Broilers Results of antioxidant capacity analyses of the serum, liver, and lungs at 21 d are shown in Table 6. The LAT environment elevated VC level and reduced T-AOC in serum (P < 0.05). The LAT environment tended to increase the levels of MDA and the amount of protein carbonylation in serum (P < 0.1). There were no differences in the activity levels of SOD and GSHPx in serum between the LAT and NAT groups (P > 0.05). Groups that were given the VC supplement tended to have increased T-AOC (P < 0.1), whereas the VC supplement decreased the activity of GSH-Px in serum (P < 0.05). There were no differences in the VC level, MDA level, protein carbonylation level, or the SOD activity in the serum between groups that were given the VC supplement and those that were not (P > 0.05). The LAT environment increased the levels of VC and protein carbonylation, and decreased T-AOC in the liver (P < 0.05). The LAT environment tended to increase the level of MDA in the liver (P < 0.1). There were
no differences in the VC level, MDA level, T-AOC, or amount of protein carbonylation in the liver between groups that were given the VC supplement and those that were not (P > 0.05). In the lungs, LAT environment increased the MDA level and the amount of protein carbonylation, and decreased T-AOC (P < 0.05). There were no differences in the VC level in the lungs between the LAT and NAT groups (P > 0.05), and no effects of the VC supplementation were observed on the VC level, MDA level, T-AOC, and amount of protein carbonylation in the lung (P > 0.05).
DISCUSSION Considering that LAT conditions are a major stressor for broilers in the winter and contribute to the high occurrence of AS in most areas, it was worthwhile to investigate the response of broilers to natural LAT conditions. The heart index value was indicative of AS because right ventricular hypertrophy is an indicator of pulmonary hypertension associated with AS (Huchzer-
Table 4. Effects of temperature and vitamin C (VC) on the blood parameters of broilers1 P-value Item HGB (g/L) 7d 14 d 21 d HCT (%) 7d 14 d 21 d RBC (× 1012/L) 7d 14 d 21 d 1LAT
LAT
LAT + VC
NAT
NAT + VC
Pooled SEM
77.6 94.7 86.7
72.2 91.0 104
71.0 79.7 90.8
75.8 81.3 96.8
2.42 4.44 2.46
0.543 * 0.613
0.903 0.828 **
0.051 0.565 0.058
Temperature
VC
Temperature × VC
0.32 0.36 0.34
0.31 0.35 0.40
0.30 0.31 0.36
0.31 0.31 0.38
0.01 0.02 0.01
0.146 * 0.946
0.908 0.854 *
0.215 0.585 0.157
2.28 2.64 2.57
2.16 2.57 2.92
2.10 2.32 2.66
2.24 2.40 2.79
0.08 0.12 0.07
0.544 0.068 0.753
0.886 0.967 **
0.108 0.519 0.127
= low ambient temperature; NAT = normal ambient temperature; HGB = hemoglobin; HCT = hematocrit; RBC = red blood cell. *P < 0.05. **P < 0.01.
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Heart index (g/g) 21 d ABWG (g) 1–7 d 8–14 d 15–21 d ADFI (g) 1–7 d 8–14 d 15–21 d FCR (g/g) 1–7 d 8–14 d 15–21 d
LAT
Pooled SEM
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YANG ET AL. Table 5. Effect of broiler age on red blood cell (RBC) count, hemoglobin (HGB) level, and hematocrit (HCT)1 Item
LAT 1012/L)
2.28b 2.64a 2.57ab 0.10 *
2.16c 2.57b 2.92a 0.10 ***
77.6b 94.7a 86.7ab 3.19 **
NAT + VC
2.10c 2.32b 2.66a 0.04 ***
72.2c 91.0b 104a 3.24 ***
0.32b 0.36a 0.34ab 0.01 *
NAT
2.24b 2.40b 2.79a 0.10 *
71.0c 79.7b 90.8a 1.70 ***
0.31b 0.35ab 0.40a 0.02 **
75.8b 81.3b 96.8a 3.70 **
0.30b 0.31b 0.36a 0.01 **
0.31b 0.31b 0.38a 0.01 **
a–cDifferent
lowercase letters in the same column mean significant differences (P < 0.05). = low ambient temperature; NAT = normal ambient temperature; VC = vitamin C. *P < 0.05. **P < 0.01. ***P < 0.001. 1LAT
meyer and De Ruyck, 1986). A previous study showed that the effect of LAT on the right ventricle was remarkable, resulting in heavier total ventricular weights and higher values for pulmonary arterial pressure (Wideman and Tackett, 2000). The heart index was generally increased with age or BW. Because LAT stress resulted in poor growth in the present experiment, the heart index values of both LAT and NAT treatments were low at 21 d. However, LAT increased heart index values compared with NAT. Therefore, these results implied that LAT caused heavier right ventricle, and it increased the possibility of developing AS.
Hypoxia has been shown to result in significant reductions in BW, which are caused by increased energy demands (Yahav and McMurtry, 2001). In the current study, hypoxia resulting from LAT conditions caused an increase in the broilers’ feed conversion ratio because the birds needed more energy to produce heat to maintain their temperature and depressed their BW gain, implying a strong LAT stress on broilers according to the growth performance. The addition of VC decreased the feed conversion ratio by reducing the ADFI during 8 to 14 d. However, there were no differences in growth performance after 14 d as a result of adding VC.
Table 6. Effects of temperature and vitamin C (VC) on the VC, total antioxidant capacity (T-AOC), malondialdehyde (MDA), protein carbonylation, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in the serum and on the VC, T-AOC, MDA, and protein carbonylation in the liver and the lungs at 21 d1 P-value
Item Serum VC (μg/mL) T-AOC (U/mL) SOD (U/mL) GSH-Px (U/mL) MDA (nmol/mL) Protein carbonylation (nmol/mL) Liver VC (μg/mg of protein) T-AOC (U/mg of protein) MDA (nmol/mg of protein) Protein carbonylation (nmol/mg of protein) Lungs VC (μg/mg of protein) T-AOC (U/mg of protein) MDA (nmol/mg of protein) Protein carbonylation (nmol/mg of protein) 1LAT
LAT
LAT + VC
NAT
NAT + VC
Pooled SEM
Temperature
VC
Temperature × VC
16.0 3.01 130 194 15.8 2.03
18.2 4.62 141 154 15.6 1.95
13.2 4.78 151 193 14.2 1.87
14.8 4.89 151 159 10.4 1.20
1.42 0.46 17.2 16.4 1.74 0.23
* * 0.372 0.909 0.070 0.063
0.207 0.082 0.766 * 0.275 0.124
0.839 0.127 0.741 0.841 0.317 0.217
2.09 0.82 1.04 4.11
2.21 0.94 0.82 3.98
1.58 0.98 0.47 2.56
1.80 1.07 0.73 3.09
0.18 0.06 0.17 0.45
* * 0.067 *
0.346 0.111 0.926 0.666
0.780 0.841 0.176 0.476
0.06 0.28 3.13 10.7
0.09 0.40 2.32 11.8
0.21 0.60 1.47 6.82
0.08 0.64 1.70 7.19
0.05 0.10 0.37 1.96
0.148 * ** *
0.339 0.434 0.439 0.708
0.098 0.670 0.174 0.847
= low ambient temperature; NAT = normal ambient temperature. *P < 0.05. **P < 0.01.
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RBC (× 7d 14 d 21 d Pooled SEM P-value HGB (g/L) 7d 14 d 21 d Pooled SEM P-value HCT (%) 7d 14 d 21 d Pooled SEM P-value
LAT + VC
EFFECTS OF LOW TEMPERATURES AND VITAMIN C ON BROILERS
Hypoxia that is induced by LAT conditions can also result in oxidation reactions. When oxidation overcomes the body’s ability to protect itself using antioxidants, oxidative stress occurs (Iqbal et al., 2002). Mujahid and Furuse (2009) have observed some oxidative stress in the brains and hearts of broilers exposed to LAT conditions. The MDA compound is an end product of the oxidative degradation of lipids and is an indicator of oxidative stress (Del Rio et al., 2005). Heat stress, which causes hypoxia, has been shown to increase MDA levels in liver tissue, both in vitro and in vivo (Feng et al., 2008). One study found that when broilers were exposed to LAT environment for 3 wk, MDA level in the plasma increased (Pan et al., 2005). Along with the MDA level, the amount of protein carbonylation in the body, which is a form of protein oxidation, is also an indicator of the body’s degree of oxidation. In agreement with Aruoma (1994), it shown that free radicals can oxidize both lipids and proteins, worsening disease conditions in broilers. In our study, LAT conditions also decreased T-AOC in the serum, liver, and lungs, but significantly increased the VC level in serum and liver. It is possible that the hypoxic conditions resulted in an inflammatory response, as the level of VC is usually higher in regions of inflammation than in areas that are not the focus of an inflammation response. In tissues and cells, many specific enzymes, such as SOD and GSH-Px, can eliminate ROS that arise from oxidative stress. The activity of GSH-Px in the mitochondria of the lungs and in the liver tissue of broilers with AS have been found to be higher than its activity in normal broilers (Iqbal et al., 2002), indicating that the amount of GSH-Px in tissues increases to eliminate ROS when necessary. However, when the amount of ROS in mitochondria exceeded the antioxidant abilities provided by SOD and GSH-Px activities, mitochondrial injury would occur (Bottje and Wideman, 1995). However, Wang et al. (2012) reported that the total SOD activity in the liver significantly decreased in broilers with AS, which indicates that AS may severely damage the mechanism for SOD production in the liver. In our experiment, the activities of SOD and GSH-Px in the serum were similar under both LAT and NAT conditions. This result might be due to the short study period (21 d) and that fact that only LAT was used as an AS-inducing factor. Vitamin C has been found to be a good scavenger of oxygen (Cort, 1974), and it can alleviate oxidative stress. However, one study showed that VC (100 mg/ kg of diet) had no significant effect on the level of SOD in the serum (Tras et al., 2000). In our study, the activity of GSH-Px in serum decreased upon addition of VC to the diet. Our results are in accordance with results from a previous study, where dietary vitamin E reduced the activity of GSH-Px in ascitic broilers as this vitamin acts also as an antioxidant in vivo (Iqbal et al., 2002). The antioxidant effects of VC were only evident as indicated by the increasing T-AOC levels in the current study. Because oxidative stress caused by
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A similar result was reported in a previous study, which revealed that adding VC (500 mg/kg) to the diet had no significant effect on growth performance (Ladmakhi et al., 1997). Shlosberg et al. (1992) reported that LAT stress caused elevated HCT, which would lead to AS. Wang et al. (2012) reported that HCT, HGB levels, and RBC counts of broilers with AS were higher than those of healthy broilers, suggesting that hypoxic conditions caused the levels of these substances to increase to fulfill tissues’ demand for oxygen. Increases in the HCT and HGB levels have been observed in broilers in LAT conditions, and blood volume values were significantly elevated at 10°C than at 20°C (Yahav et al., 1997; Wideman et al., 1998; Wideman and Tackett, 2000; Luger et al., 2001). In the present study, LAT conditions markedly increased the HCT and HGB levels at 14 d, suggesting that LAT conditions had elevated the possibility of birds developing AS. Even though many studies have investigated the levels of these blood parameters related to AS, there are few reports about the effect of VC on blood parameters of broilers. In calves experiencing heat stress, one study showed that a VC supplementation (20 g/d) decreased their RBC counts, HCT, and HGB levels (Kim et al., 2012). When investigating these blood parameters in broilers, Tras et al. (2000) found that VC (100 mg/ kg of diet) had no significant effect. In our study, RBC counts, HCT, and HGB levels in broilers that were given a VC supplement increased at 21 d, especially for broilers in LAT conditions. This was a compensation mechanism. Because in some condition, the lower HCT level indicated less erythrocytes in blood volume, and it induced a greater risk of hypoxia. Broilers with lower HCT level had significantly higher mortality other than mortality caused by AS (Shlosberg and Bellaiche, 1996). The increase of RBC counts, HGB, and HCT levels due to VC addition were not anticipated; therefore, the mechanism was not investigated in this study. These results provide further evidence that the increase in RBC counts, HCT, and HGB levels induced by LAT conditions resulted from the increased need for oxygen in broilers. Hypoxia has been shown to increase the amount of reactive oxygen species (ROS) in cells and mitochondria and disturb the oxidation-reduction system in the body, as well as stabilize the hypoxia-inducible factor, leading to vascular remodeling, which is the foundation of AS (Chandel et al., 1998, 2000). Free radicals or ROS can result in lipid and protein peroxidation, and if it is not reduced by antioxidant protective mechanisms, tissues would be damaged. Supplementation with VC has been found to alleviate the oxidative damage induced by ROS and therefore allow cells to maintain their proper redox state (Blagojeviü, 2007). Broilers living in conditions of low ventilation have been reported as having low levels of VC in their liver and lungs, which suggests that VC is consumed in the oxidation reactions caused by hypoxia (Enkvetchakul et al., 1993).
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it needs further study when broilers are reared under LAT conditions.
ACKNOWLEDGMENTS We thank Todd. J. Applegate and Chen Xi (Animal Science Department, Purdue University, West Lafayette, IN) for their role in revising the paper. This research was supported by grants from the National Natural Science Foundation of China (31101733).
REFERENCES Aengwanich, W., and S. Simaraks. 2004. Pathology of heart, lung, liver and kidney in broilers under chronic heat stress. Pathology 26:418. Aruoma, O. I. 1994. Free radicals and antioxidant strategies in sports. J. Nutr. Biochem. 5:370–381. Bautista-Ortega, J., and C. A. Ruiz-Feria. 2010. l-Arginine and antioxidant vitamins E and C improve the cardiovascular performance of broiler chickens grown under chronic hypobaric hypoxia. Poult. Sci. 89:2141–2146. Blagojeviü, D. P. 2007. Antioxidant systems in supporting environmental and programmed adaptations to low temperatures. Cryo Letters 28:137–150. Bottje, W. G., and R. F. Wideman. 1995. Potential role of free radicals in the pathogenesis of pulmonary hypertension syndrome. Poult. Avian Biol. Rev. 6:211–231. Carr, A. C., B. Z. Zhu, and B. Frei. 2000. Potential antiatherogenic mechanisms of ascorbate (vitamin C) and α-tocopherol (vitamin E). Circ. Res. 87:349–354. Chandel, N. S., E. Maltepe, E. Goldwasser, C. E. Mathieu, M. C. Simon, and P. T. Schumacker. 1998. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc. Natl. Acad. Sci. USA 95:11715–11720. Chandel, N. S., D. S. McClintock, C. E. Feliciano, T. M. Wood, J. A. Melendez, A. M. Rodriguez, and P. T. Schumacker. 2000. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1α during hypoxia. J. Biol. Chem. 275:25130–25138. Cort, W. M. 1974. Antioxidant activity of tocopherols, ascorbyl palmitate, and ascorbic acid and their mode of action. J. Am. Oil Chem. Soc. 51:321–325. Daneshyar, M., H. Kermanshahi, and A. Golian. 2009. Changes of biochemical parameters and enzyme activities in broiler chickens with cold-induced ascites. Poult. Sci. 88:106–110. Decuypere, E., C. Vega, T. Bartha, J. Buyse, J. Zoons, and G. A. A. Albers. 1994. Increased sensitivity to triiodothyronine (T3) of broiler lines with a high susceptibility for ascites. Br. Poult. Sci. 35:287–297. Del Rio, D., A. J. Stewart, and N. Pellegrini. 2005. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr. Metab. Cardiovasc. Dis. 15:316–328. Enkvetchakul, B., W. Bottje, N. Anthony, R. Moore, and W. Huff. 1993. Compromised antioxidant status associated with ascites in broilers. Poult. Sci. 72:2272–2280. Feng, J., M. Zhang, S. Zheng, P. Xie, and A. Ma. 2008. Effects of high temperature on multiple parameters of broilers in vitro and in vivo. Poult. Sci. 87:2133–2139. Guney, M., B. Oral, H. Demirin, N. Karahan, T. Mungan, and N. Delibas. 2007. Protective effects of vitamins C and E against endometrial damage and oxidative stress in fluoride intoxication. Clin. Exp. Pharmacol. Physiol. 34:467–474. Huchzermeyer, F. W., and A. M. De Ruyck. 1986. Pulmonary hypertension syndrome associated with ascites in broilers. Vet. Rec. 119:94. Iqbal, M., D. Cawthon, K. Beers, R. F. Wideman, and W. G. Bottje. 2002. Antioxidant enzyme activities and mitochondrial fatty
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LAT conditions was severe, supplemental VC did not improve the antioxidant capacity. Vitamin C generally acted as a co-antioxidant that functioned by cooperating with other antioxidants, for instance, with vitamin E. Vitamin C was shown to restore the antioxidant capability of vitamin E by converting the tocopherol radical back to its reduced state (Witting and Horwitt, 1964; Carr et al., 2000; Guney et al., 2007). Vitamin C cooperating with vitamin E can become a scavenger of ROS to protect NO, which was a substance maintaining pulmonary vasodilatation (Ruiz-Feria, 2009; Bautista-Ortega and Ruiz-Feria, 2010). In our study, vitamin E was not added to the basal diet, and soybean oil was included, which was apt to be oxidized. Vitamin C with inappropriate supplementation can also become a pro-oxidative agent by reducing Fe3+ to Fe2+, which then generated free radicals by interacting with Cu2+ (Bottje and Wideman, 1995). This suggests that the proper cooperative substance and the additive amount of VC should be continually explored when the broilers are reared under LAT conditions. The change in antioxidant capacity (T-AOC) observed in this study indicated that hypoxia, induced by LAT conditions, had a marked effect on the different tissues, especially the lungs. Edema and hyperemia of the lungs have been observed in broilers experiencing chronic heat stress, which causes hypoxia, and hemorrhaging was observed in their alveoli, indicating that lung lesions are common consequences of hypoxia (Aengwanich and Simaraks, 2004). When AS is present, cell growth and migration of pulmonary arterioles have been found (Nain et al., 2009); pulmonary arterioles become more muscular, evident through observation of thicker media and cellular proliferation in the intima, which results in pulmonary hypertension (Xiang et al., 2002). In addition, a study by Iqbal et al. (2001) showed that hydrogen peroxide, another biomarker of oxidative stress, was overproduced in the lung mitochondria of broilers experiencing pulmonary hypertension syndrome (Iqbal et al., 2001). Similarly, the present study showed that LAT conditions worsened the oxidative stress in broilers, as evidenced by changes in the levels of T-AOC, MDA, and protein carbonylation. Although it has been reported that VC (500 mg/kg of diet) can alleviate AS by reducing the muscularization of the pulmonary arterioles (Xiang et al., 2002), it had little effect on maintaining proper lung function in the present study. In conclusion, the natural LAT scheme employed in this experiment caused oxidative stress of broilers over a 21-d period, as shown by the significant increase in HCT, poor growth, and low antioxidant capacities. Although the LAT conditions failed to induce large-scale incidence of AS, it can be used as a model for future studies that focusing on the mechanisms by which hypoxia leads to oxidative stress. The effect of the dietary VC supplementation on the antioxidant capacity of 21-d-old broilers was not significant, indicating that
EFFECTS OF LOW TEMPERATURES AND VITAMIN C ON BROILERS
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