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Contemplation Upon Nano Red Selenium and Sodium Selenite on Antioxidant Enzymes in Quail Under Heat Stress

Published: July 23, 2019
By: R. Ghazanfarpoor, E. Talebi and A. Abedi / Young Researchers and Elite Club, Darab Branch, Islamic Azad University, Darab, Fars, Iran.

This study was designed to investigate the effects of Nano-Selenium and Sodium Selenite on some blood biochemical parameters in Japanese quail under heat stress condition. In this investigation, 300 male Japanese quails were used which the chickens were raised to 21 days in same condition. The experiment was performed in a randomized complete block design (RCBD) using 3 treatments and 5 replicates with 20 chicks per each replicate. Quails in the control groups were used the basal diet (without selenium supplement) and the first experimental group admitted basal diet enriched with 0.2 mg/kg Nano-Selenium subsequently the second experimental group employed basal diet with 0.2 mg/kg Sodium Selenite. The chickens daily were included under heat stress condition for 8 h at 34± 0.5oC. The results revealed that glutathione peroxidase and superoxide dismutase activity increased significantly in second experimental group using selenium (p<0.05). In the present study, superoxide dismutase activity was significantly increased after adding 0.2 mg sodium selenite and nano-selenium (p<0.05).

Keywords: Nano-Selenium, Sodium Selenite, Glutathione Peroxidase, Superoxide Dismutase, Heat stress.

Nanotechnology is regarded as the ability to manipulate measure, manufacture and make predictions at the scale of 1–200 nm. At the nanometer dimension, materials revealed novel properties, different to those of both, the isolated atom and bulk material that the properties depending largely on the size of the particles from which the material is made.
The environmental stress can decrease production and all other important economical traits in animal husbandry. The stress is caused by free radicals and reactive oxygen species. Detrimental effects caused by reactive oxygen species occur as a consequence of an imbalance between the formation and inactivation of these species. Oxidative damage may be involved in the pathogenesis of major diseases such as cancer, atherosclerosis, and certain neurological disorders as well as caused by environmental stresses. The free radicals are atoms or molecules which they are active at the last atomic layer due to their strong affinity with their surrounding molecules (Alexanra et al., 2002). Animals need trace mineral elements in the diet to maintain health and proper essential biochemical and physiological function. The relationship between selenium and energy metabolism, increase feed efficiency, as well as improves reproductive and immune systems (Tamilson et al., 2008).
Selenium is important to human and animal health and it has many biological functions in living organisms, involved in the regulation of antioxidant mechanisms (Rayman, 2000). Approximately, 40 to 50 percent of the total body selenium exists in glutathione peroxidase. In the form of selenocysteine, the 21st amino acid, Se functions as a redox center of an array of selenoproteins (Copeland, 2003; Driscoll and Copeland, 2003), some of which have important enzymic functions for the redox homoeostasis, such as glutathione peroxidase (GPx) (de Haan et al., 2003; Miyamoto et al., 2003), phospholipid hydroperoxide glutathione peroxidase (PHGPx) (Imai and Nakagawa, 2003; Nakagawa and Imai, 2000) and thioredoxin reductase (TrxR) (Arner and Holmgren, 2000; Becker et al., 2000). Selenium in body increases the amount of enzyme activity about 100 to 1000 times (Burk, 2002). Superoxide anion is the first free radical which derived from oxygen and it is turned and neutral to the oxygen and hydrogen peroxide by superoxide dismutase. Subsequently, hydrogen peroxide converted to water by glutathione peroxidase (Mates, 1999). Glutathione peroxidase, superoxide dismutase and catalase, as well as non-enzymatic molecules such as vitamins C, E and A, uric acid, bilirubin, protect lipids and nucleic acids from oxidative damage which play an important role in protecting the cell membrane (Michiels et al., 1994). The important antioxidant enzymes characteristic is the induction of them in oxidative stress conditions. Thus, cells induction caused by oxidative stress and an increase in the activity of antioxidant enzymes exhibit the cell or tissue adaptation to stress condition (John et al., 2001). Several studies also have shown low levels or absence of lipid peroxidation and antioxidant enzymes may reflect a protective effect (Nordberg and Arner, 2001). This study was conducted to examine Nano Red Selenium and Sodium Selenite effects on some antioxidant enzymes in quail under heat stress condition.
Materials and methods
Animals and experimental conditions:
In this investigation, 300 Japanese quail chicks were used and the chickens were raised to 21 days in the same condition. The chickens were kept in cages 50 × 90 cm. The experiment was performed in a randomized complete block design (RCBD) with 3 treatments and 5 replicates using 20 chicks per replicate. The chickens daily were included under heat stress condition for 8 h at 34± 0.5oC.
Preparation of Nano Red Selenium:
The nano red selenium was prepared by Zhang et al. (2004) method. Ingredients for preparation of the nano red selenium were including ascorbic acid and SeO2. The ascorbic acid solution was added into the aqueous solution of SeO2 to initiate the reaction. After the addition of ascorbic acid, nano red selenium particles began to formation by color changing from colorless to red. The nano red selenium particle size was obtained in range of 80 to 200 nm which determined by Scanning Electron Microscopy. Sodium Selenite was purchased from Sigma Corporation of America.
During the first 21 days, quail chickens were fed basal diet without selenium supplement. The basal diet (Table 1) was prepared according to National Research Council (NRC). The treatments included 1: basal diet (without selenium supplement), 2: basal diet + 0.2 mg/kg sodium selenite, 3: basal diet + 0.2 mg of selenium nanoparticles.
Table 1: Feed and chemical composition of basal diet *The mineral supplement was free of selenium. 
Contemplation Upon Nano Red Selenium and Sodium Selenite on Antioxidant Enzymes in Quail Under Heat Stress - Image 1
Two chicks per pen were weighted close to the average weight per cage. The blood samples were taken under the wing vein. Blood samples collected in tubes containing anticoagulant (heparin). After hematocrit and hemoglobin determination using a kit from Randox (England), superoxide dismutase and glutathione peroxidase activity were measured in red blood cells.
Measurement of Glutathione peroxidase:
Glutathione peroxidase (glutathione peroxidase) catalyzes the reduction of hydroperoxides, including hydrogen peroxide, by reduced glutathione and functions to protect the cell from oxidative damage. With the exception of phospholipid-hydroperoxide glutathione peroxidase, a monomer, all of the glutathione peroxidase enzymes are tetramers of four identical subunits. GPX is catalyzed oxidation of glutathione (GSH) by Komen hydroperoxides. In the presence of glutathione reductase (GR) and NADPH, the oxidized glutathione (GSSG) immediately reacts with the recovery in output and the oxidation of NADPH into NADP +. For glutathione peroxidase determination, absorbance is measured at 340 nm.
Measurement of superoxide dismutase:
The amount of SOD present in cellular and extracellular environments is crucial for the prevention of diseases linked to oxidative stress. Superoxide dismutases (SODs) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and thus form a crucial part of the cellular antioxidant defense mechanism. In this method, using xanthine oxidase is able to react with 2- (4–nitro phenol) -3-5- phenol tetrazolium chloride to produce red formazan at wavelength of 505 nm. For determination of enzyme activity in presence of SOD enzyme, superoxide radicals converted to hydrogen peroxide and O2 to produce red formazan and it is inhibited to produce red.

Statistical analysis:
The data were analyzed by following statistical model: 
Yij = µ + Ti + Rj + eij
Yij: each observation μ: overall mean,
Ti: treatment effect,
Rj: block effect (cage floor)
eij: error
The results were analyzed using SAS software (version 14) through two-way analysis and Duncan test.
Results and Discussion:
According to antioxidant role of selenium and antioxidant enzymes induced in stress conditions, the effect of sodium selenite and nano selenium was compared on antioxidant enzymes activity. The result revealed that the addition of 0.2 mg/kg nano selenium and sodium selenite increased significantly glutathione peroxidase activity than the control group (p<0.05) (Figure 1). Nano selenium on the activity of the enzyme was more effective compared to sodium selenite, but it was not significant (p>0.05). Payen and Southern (2005) reported that selenium has positive effect on blood glutathione peroxidase activity in poultry. Increased blood levels of glutathione peroxidase in broiler when there is a sufficient amount of selenium in diet play an important protective response against oxidative damage which caused by heat stress (Altan et al., 2003). Zeinali et al. (1388) and Ramazani et al. (1390) exhibited that addition of 0.3 mg/kg selenium to diet can increase glutathione peroxidase activity under heat stress condition. Camel et al. (2003) showed positive effects of selenium supplement on glutathione peroxidase activity in chicken’s blood under heat stress conditions. Recent research has also reported that using of Nano selenium in broiler diet caused a significant increase in glutathione peroxidase activity and free radical scavenging (Cai et al., 2012). Paton et al. (2002) reported that addition of selenium to diet with selenium deficiency can increase GPx activity. Addition of selenium to the diet is caused in higher retention of vitamin E and vitamin E in plasma also reduce production of Hydro peroxidase which decreased glutathione to protect cells by reducing the need and this could be the reason for increased glutathione peroxidase (Lin et al., 2006). Contor et al. (1975) reported that usage of control diet and diets containing organic or inorganic selenium sources in amount of 0.2 mg/kg were no significant effect on glutathione peroxidase activity. The comparison between nano red selenium and sodium selenite on glutathione peroxidase activity and antioxidant systems demonstrated that nano red selenium is suitable for increasing glutathione peroxidase activity in quail under heat stress. Xu et al. (2003) showed that the most appropriate form of selenium is nano-selenium, selenomethionine and sodium selenite.  Another part of the defensive system of enzymatic antioxidant is defense system of cells against oxidative stress. The superoxide dismutase is a rapid removal of free radicals. Kucuk et al. (2003) stated that superoxide dismutase is the first line of cellular defense against toxic free radicals caused by free radicals. Thus, superoxide dismutase is an important enzyme for prevention of cell lipid peroxidation and production of free radicals such as O2 and HO in blood and tissues. Antioxidant such as selenium, vit E etc. increase antioxidant enzyme levels in the blood of chickens under stress condition.
Contemplation Upon Nano Red Selenium and Sodium Selenite on Antioxidant Enzymes in Quail Under Heat Stress - Image 2
Figure 1: Effect of Nano Selenium and Sodium Selenite on GPx in quial under heat stress condition.
In the present study, superoxide dismutase activity was significantly increased after adding 0.2 mg sodium selenite and nano-selenium (p<0.05) (Figure 2). Zeinali et al. (2009) and Ramazani et al. (2011) also reported significant effect of selenium supplementation on serum superoxide dismutase levels in broilers under heat stress. Angkananporn and kijiparkorn (2003) revealed that chickens fed with selenium supplementation showed significantly higher level of SOD (superoxide dismutase activity) compared to the control group, which is consistent with the results of this study. Furthermore, Pirseljein et al. (2006) reported that dietary supplementation of organic selenium had different effect on superoxide dismutase activity in broiler. Researchers have shown that superoxide anion is the first oxygen-derived free radicals to oxygen and hydrogen peroxide by SOD. Subsequently, hydrogen peroxide converts to water by glutathione peroxidase (Mates, 1999). 
Contemplation Upon Nano Red Selenium and Sodium Selenite on Antioxidant Enzymes in Quail Under Heat Stress - Image 3
Figure 2: Effect of Nano Selenium and Sodium Selenite on SOD in quail under heat stress condition.
Glutathione peroxidase and superoxide dismutase activity is measured in whole blood and red blood cells and the enzyme compared with hematocrit and hemoglobin and discussion on hematocrit and hemoglobin is important for this type research. Experiments showed that selenium deficiency is associated with anemia due to reduced hemoglobin. Selenium protects the hemoglobin and iron absorption from the intestine is inhibited (Pavlik et al., 2012). The results of this study indicated that the usage of selenium supplements increase blood hematocrit compare to control group (Figure 3), but this increase was not significant (p>0.05). Between the two groups using sodium selenite and Nano-Selenium was also no difference in percentage of hematocrit.
Contemplation Upon Nano Red Selenium and Sodium Selenite on Antioxidant Enzymes in Quail Under Heat Stress - Image 4
Figure 3: Effect of Nano Selenium and Sodium Selenite on hematocrit percentage in quail under heat stress condition.
The Selenium supplements increased significantly hemoglobin compared to control group (p<0.05) (Figure 4) and the use of nano-selenium treatment compare to sodium selenite exhibited higher hemoglobin but the difference was not significant. Mohri et al. (2005) reported that cows injected with selenium were significantly increased hematocrit, hemoglobin and white blood cells in calves which are consistent with the results of our study. According to antioxidant protective effect of selenium reduced excretion of electrolytes such as iron which is the main component of hemoglobin under heat stress condition and subsequently hemoglobin will be increase. Therefore, selenium is increased hematocrit and hemoglobin that increase in hemoglobin and hematocrit levels of selenium can be used to protect cell membranes and intracellular tissues by antioxidant effects of this element. This is likely to increase the life span of red blood cells.
Contemplation Upon Nano Red Selenium and Sodium Selenite on Antioxidant Enzymes in Quail Under Heat Stress - Image 5
Figure 4: Effect of Nano Selenium and Sodium Selenite on hemoglobin in quail under heat stress condition It is clear from the results that nano-selenium supplementation increases glutathione peroxidase enzyme and hemoglobin compare to sodium selenite. This is probably due to less toxicity, bioavailability, higher absorption and catalytic efficiency of selenium nanoparticles (Zhang et al., 2001).
This article was originally published in International Journal of Science Inventions Today, 2014, 3(6), 556-565.

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Dr. Ebrahim Talebi
Juarez Donzele
Universidade Federal de Viçosa - UFV
6 de abril de 2020

Dr.  Ebrahim, very good work. Based on your knowledge of the subject at hand, I would like your opinion on whether, in thermal stress, selenium would have any effect on the expression of any of the heat shock proteins (HSP) and / or the hormone Corticosterone. If so, this or these factors could have contributed to the results obtained in your work.

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