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Journal of Animal Science - Animal Production

Effects of methionine supplementation on the redox state of acute heat stress–exposed quails1


This article in JAS

  1. Vol. 92 No. 2, p. 806-815
    Received: June 26, 2013
    Accepted: Nov 25, 2013
    Published: November 24, 2014

    2 Corresponding author(s):

  1. A. P. Del Vesco*,
  2. E. Gasparino 2,
  3. D. O. Grieser*,
  4. V. Zancanela*,
  5. F. R. S. Gasparin,
  6. J. Constantin and
  7. A. R. Oliveira Neto
  1. Department of Animal Science, Universidade Estadual de Maringá – UEM – Maringá, Paraná, Brazil
    Department of Biochemistry, Universidade Estadual de Maringá – UEM– Maringá, Paraná, Brazil
    Evonik-Degussa, Brazil


The aims of the present study were to evaluate the possible effects of heat stress (HS) on H2O2 production and to evaluate whether methionine supplementation (MS) could mitigate the deleterious effects on cell metabolism and the redox state induced by oxidative stress. Meat quails (Coturnix coturnix coturnix) were fed a diet that either met the nutritional demands for methionine or did not meet this demand (methionine deficient [MD] diet) for 7 d. The animals were either kept at a thermal comfort temperature (25°C) or exposed to HS (38°C for 24 h, starting on the sixth day). Heat stress induced decreased food intake (P = 0.0140), decreased daily weight gain (P < 0.0001), and increased water intake (P = 0.0211). A higher rate of H2O2 production was observed in HS animals (0.0802 vs. 0.0692 nmol of reactive oxygen species [ROS] produced per minute per milligram of protein; P = 0.0042) and in animals fed with the MD diet (0.0808 vs. 0.0686 nmol of ROS produced per minute per milligram of protein; P = 0.0020). We observed effects of the interaction between diet and the environment on the activities of glutathione peroxidase (GP-x) and catalase (P = 0.0392 and P < 0.0001, respectively). Heat stress induced higher levels of GP-x activity in animals on the MS diet and higher catalase activity in animals on the MD diet. Glutathione (GSH) levels were higher in animals on the MS diet (P = 0.0273) and in animals that were kept in thermal comfort (P = 0.0018). The thiobarbituric acid reactive substances level was higher in HS animals fed with the MD diet (P = 0.0386). Significant effects of the interaction between supplementation and environment were observed on uric acid concentration levels, which were higher in HS animals fed the MS diet (P = 0.008), and on creatine kinase activity levels, which were lower in HS animals fed the MD diet (1,620.33 units/L; P = 0.0442). Our results suggest that under HS conditions, in which H2O2 production is increased, MS was able to mitigate ROS-induced damage, possibly by increasing the activities of antioxidant elements such as GSH, GPx activity, and uric acid concentration, which were present in higher levels in animals that were subjected to HS and fed the MS diet.


Reactive oxygen species (ROS) are normally produced in cellular biological processes. At increased levels, ROS are frequently associated with events such as apoptosis, protein oxidation, lipid peroxidation, and mitochondrial DNA damage (Moustafa et al., 2004; Lee and Wei, 2005). An imbalance between the production and elimination of ROS in an organism leads to a state known as oxidative stress. Several studies have linked heat stress (HS) with oxidative stress (Mujahid et al., 2009, 2005), and the effects of HS are possibly due to an acceleration in the rate of ROS formation and/or an increase in ROS reactivity (Bai et al., 2003). Despite extensive studies on the subject, the mechanism through which high temperatures influence the cell redox state is not yet fully clear. The decreased expression of uncoupling proteins (UCP) that occurs in birds exposed to HS (Del Vesco and Gasparino, 2012) might be one such mechanism involved in ROS production because proteins such as UCP and adenine nucleotide translocases could depolarize mitochondrial internal membranes, thus exerting (along with the other remaining factors) physiological control on oxidative phosphorylation (Sack, 2006).

The defenses of organisms against ROS may be mediated by nonenzymatic or enzymatic antioxidants, mainly represented by the enzymes superoxide dismutase and catalase, and the glutathione (GSH) defense system (Abrashev et al., 2008).

Glutathione is involved in various biological actions, including protection against toxic compounds, the reduction of disulfide bonds in proteins, service as a precursor for DNA synthesis, service as a cysteine pool and, above all, protection against free radicals (Morand et al., 1997). Glutathione is biosynthesized in the majority of tissues from 3 precursor AA, one of which, cysteine, can be synthesized from a methionine precursor via the homocysteine transsulfuration pathway (Shoveller et al., 2005).

Estimates have suggested that approximately 50% of GSH is generated from homocysteine and that under oxidative stress conditions, the production rate increases through stimulation of the transsulfuration pathway via the increased expression and activity of cistathionine β-synthase and the inhibition of methionine synthase, which are the enzymes responsible for cysteine and methionine synthesis, respectively (Mosharov et al., 2000; Persa et al., 2004). In addition to playing an important role in GSH synthesis (reviewed by Stipanuk, 2004), some studies have shown the direct protective effects of methionine on oxidative stress (Stadtman et al., 2002; Moskovitz et al., 2001; Levine et al., 2000).

The present study was performed to test the hypothesis that HS induces oxidative stress and that under stress conditions, methionine supplementation (MS) may contribute to antioxidative systems, thus reducing oxidative stress damage. Therefore, the present study aimed to evaluate the effects of HS and MS on meat quails performance and to evaluate the role of HS in H2O2 production and whether MS could mitigate the deleterious effects of oxidative stress on the metabolism and redox state in birds.


The procedures in this experiment were approved by the Committee on Animal Care of the Universidade Estadual de Maringá – Brazil.

Experimental Design and Animals

Sixty-four male meat quails (Coturnix coturnix coturnix) were raised in floor pens with wood shavings as litter under thermal comfort temperature until 35 d of age and were fed a balanced diet to meet their nutritional demands (Rostagno et al., 2011). After 35 d, the animals were divided into 4 groups (n = 16 per group) and transferred to individual cages in temperature-controlled rooms (20 m2; room 1 and 2: 2 groups per room) where they remained for 7 d while adapting to the thermal comfort temperature: 25 ± 0.9°C with 60 ± 1.2% relative humidity. The temperature chosen for thermal comfort followed the recommendations of Pinto et al. (2003). In each room, 1 group received feed that was calculated to meet the nutritional demand for methionine, according to Rostagno et al. (2011), and the other group received feed without methionine supplementation (methionine deficient [MD] diet; Table 1). Animals in room 1 were kept at thermal comfort during the 7 d of evaluation while animals in room 2 were kept at thermal comfort for 6 d. At the end of the sixth day, these animals were subjected to HS of 38°C for 24 h. All animals were sacrificed by cervical dislocation at the end of the seventh day. Water and feed intake were measured during the full thermal comfort period and the 24 h of stress period.

View Full Table | Close Full ViewTable 1.

Diet components and calculated and analyzed contents of the experimental diets (as-fed basis)

Experimental diet1
Ingredient MS MD
Corn 7.8% CP 58.25 58.40
Soy bean meal 46.7% CP 35.40 35.40
Soy oil 2.40 2.50
Salt 0.40 0.40
Calcareous 38% 1.34 1.34
Dicalcium phosphate 20% 1.33 1.33
dl-Methionine 99% 0.27
l-Lysine HCl 78% 0.15 0.15
l-Treonine 78% 0.06 0.06
Premix2 0.40 0.4
Total 100.00 100.00
Composition analysis, %
    CP 21.16 21.02
    Lysine digestible 1.15 1.15
    Met + Cis digestible 0.84 0.57
    Treonine digestible 0.78 0.78
    Triptophane digestible 0.23 0.23
    Valine digestible 0.90 0.90
    Isoleucine digestible 0.83 0.83
    Arginine digestible 1.33 1.33
Composition calculated,%
    Ca 0.90 0.90
    P 0.41 0.41
    Na 0.18 0.18
    AME,3 kcal/kg 2,999.06 3,000.08
1MD = methionine deficient; MS = methionine supplementation.
2Supplied by kilogram of diet: retinyl-acetate, 3.44 mg; cholecalciferol, 50 μg; DL-α-tocopherol, 15 mg; thiamine, 1.63 mg; riboflavin, 4.9 mg; pyridoxine, 3.26 mg; cyanocobalamin, 12 μg; D-pantothenic acid, 9.8 mg; D-biotin, 0.1 mg; menadione, 2.4 mg; folic acid, 0.82 mg; niacinamide, 35 mg; selenium, 0.2 mg; iron, 35 mg; copper, 8 mg; manganese, 60 mg; Zn, 50 mg; I, 1 mg; choline, 650 mg; salinomycin, 60 mg; avilamycin, 5 mg; and butyl hydroxy toluene, 80 mg.
3AME = apparent metabolizable energy.

Body Weight and Sample Collection

To calculate the weight gain of the quails under thermal comfort and stress conditions, the specimens were weighed individually at the beginning and the end of the thermal comfort and stress periods, respectively.

The livers, legs, breasts, and abdominal fat of 6 specimens from each treatment group (comfort MS, comfort MD, stress MS, and stress MD) were weighed to obtain the proportional organ weights, which were calculated as (organ weight/bird weight) × 100. Additionally, blood was collected from these animals for uric acid and creatinine content and plasma creatine kinase (CK) activity analysis (n = 6). Blood was collected from the jugular veins into heparin tubes and was kept on ice. After centrifugation (3.024 × g for 10 min at 4°C), plasma was collected and stored at –20°C until analyzed.

Uric acid, creatinine, and CK activity analyses were performed according to colorimetric methods with the following kits: uric acid MS 80022230171, creatinine-PP MS80022230066, and CK-NAC-PP MS 80022230088, respectively, following the manufacturer’s recommendations (Gold Analisa, Belo Horizonte, Minas Gerais, Brazil). One unit of CK activity was defined as the amount of enzyme needed to convert 1 mmol of creatine into creatine phosphate per minute at 37°C, pH 9.0.

Six quail livers from each treatment group were collected for thiobarbituric acid reactive substances (TBARS), GSH, catalase activity, and glutathione peroxidase (GP-x) activity analysis. All collected samples used in biochemical analyses were frozen in liquid nitrogen and stored at –80°C until analyzed.

The production of ROS was evaluated in mitochondria isolated from the livers of 4 quails from each treatment group.

Reactive Oxygen Species Production

Four quails from each treatment group were sacrificed to analyze ROS production. The livers were collected for mitochondrial isolation and subsequent analysis of mitochondrial ROS content. For mitochondrial isolation, the livers were placed in a cooled Becker container with isolation medium (0.2 M mannitol, 0.075 M sucrose, 2.0 mM Tris HCl, pH 7.4, 0.2 mM EDTA, 100 μM phenylmethylsulphonylfluoride, and 50 mg fatty acid–free bovine serum albumin; all the products were obtained from Sigma-Aldrich, St. Louis, MO) and sliced with scissors. The livers and isolation medium were transferred into a Dounce homogenizer. The homogenates were filtered and subjected to 2 sequential centrifugations at 536 × g and 7,080 × g for 10 min at -4°C each each. The precipitates were washed twice by centrifugation at 6,392 × g at -4°C. Mitochondria were homogenized in the same medium at a protein concentration of 80 to 100 mg/mL (Bracht et al., 2003).

The protein content of the subcellular fractions was determined according to the method described by Lowry et al. (1951) with bovine albumin as a standard and a spectrophotometer set for 700 nm wavelength.

The mitochondrial ROS production level was estimated by measuring the linear increase in fluorescence. Hydrogen peroxide induces dichlorofluorescein diacetate (DCFH-DA) oxidation that generates a fluorescent compound (2′-7′ dichlorofluorescein) in the presence of horseradish peroxidase (HRP; Zaccagnino et al., 2009).

Intact mitochondria (10 μL containing approximately 0.8 mg protein) were added to 2 mL of 250 mM Mannitol and 10 mM HEPES, pH 7.2, buffer with 1.36 μM DCFH-DA and succinate (10 mM) plus rothenone (10 μM) or succinate (10 mM) plus rothenone plus antimycin (15 μM). The reactions were started by the addition of 0.4 μM HRP, and the fluorescence levels were measured every minute for 10 min (Siqueira et al., 2005; all the products were obtained from Sigma-Aldrich). The assays were performed in a spectrofluorometer with stirring. The results are expressed as the nanomoles of produced ROS per minute per milligram of protein.

Enzymatic Analysis

Six quails from each treatment group were used for GP-x activity analysis. Immediately after slaughter, the livers were collected from the animals, pressed, and stored in liquid nitrogen until analyzed. For enzymatic activity analysis, the samples were weighed and crushed in liquid nitrogen with a mortar and pestle. The crushed samples were transferred to cooled test tubes, to which 10% of the sample weight of iced phosphate buffer (0.1 M K2HPO4 and KH2PO4, pH 7.4) was added. The samples were homogenized with a Potter-Elvehjem homogenizer. The liver homogenate was centrifuged at 6,392 × g for 10 min at -4°C. and enzymatic activities were determined in the supernatants. The protein contents were determined according to Lowry et al. (1951) with bovine serum albumin as the standard.

Glutathione peroxidase activity was determined with H2O2 according to the method described by Paglia and Valentine (1967). The activity of the enzyme was determined by the amount of nicotinamide adenine dinucleotide phosphate (NADPH) oxidized that was detected using the spectrophotometer at a wavelength of 340 nm to measure the fluorescent signal emitted by the consumed NADPH. The fluorescent signal was measured every 10 s for 50 s. The consumed NADPH was measured based on the decay in absorbance over the evaluated time period.

For the reactions, 350 μL of 171 mM potassium phosphate buffer, 250 μL of 6 mM GSH, 300 μL of 0.9 mM NADPH, 10 μL of 2 units (U)/mL glutathione reductase (all the products were obtained from Sigma-Aldrich), 40 μL of supernatant, and 520 μL of deionized H2O were combined. The solution was homogenized, and 30 μL of 6 mM H2O2 were subsequently added. The fluorescence values were recorded every 10 s for 50 s. The activity was expressed as the nanomoles of oxidized NADPH per milligram of protein per minute.

Catalase activity was determined by the rate of H2O2 reduction into water and oxygen, according to the method proposed by Beers and Sizer (1952). The results were expressed as units per milligrams of protein.

Lipid Peroxidation and Glutathione Determination

Lipid peroxidation was measured in the liver homogenates by evaluating the TBARS with malondialdehyde (1,1,3–3-tetrametoxipropane), according to Iguchi et al. (1993). The results were expressed as nanomoles of TBARS per milligram of protein.

For GSH analysis, the liver fractions were homogenized with perchloric acid and centrifuged at 15,000 × g for 1 min at 4°C. 5,5′-dithiobis-2-nitrobenzoate was added to the supernatants, and the reactions were performed according to the method described by Teare et al. (1993). The results are expressed as micrograms per milligram of protein.

Protein concentrations were determined according to the method described by Lowry et al. (1951) with bovine serum albumin as the standard.

Statistical Analysis

The results are expressed as averages and standard deviations. The UNIVARIATE procedure was applied to evaluate data normality. The experiment was conducted in a completely randomized factorial design, with 2 environments (thermal comfort and HS) and 2 methionine supplementation hypotheses (MS and MD). The averages were compared using Tukey’s test (P < 0.05; SAS Inst. Inc., Cary, NC).


Water and Feed Consumption, Daily Weight Gain, and Proportional Organ Weight

Figure 1 shows the water and feed intakes that were recorded for the 2 diets and 2 environments being studied. Methionine supplementation was not found to affect either factor; however, HS significantly increased water intake (P = 0.0211) and decreased feed intake (P = 0.0140).

Figure 1.
Figure 1.

Daily water and feed intake of quails in thermal comfort or heat stress (HS) and fed with methionine deficient (MD) or methionine supplementation (MS) diet. The different letters between the treatment groups represent a significant difference (P < 0.05).


The effects of supplementation and high temperature on the daily weight gain (DWG) and the proportional weights of the liver, abdominal fat, breast, and legs are shown in Table 2.

View Full Table | Close Full ViewTable 2.

Daily weight gain (DWG) and proportional weight of organs and muscles

Diet - Environment Diet × Environment DWG, g
Proportional weight (%)
Abdominal fat
Mean SD Mean SD Mean SD Mean SD Mean SD
MS1 Comfort 1.61 0.079 1.10 0.19 2.21 0.50 25.92 1.79 14.96 0.44
Stress 0.31 0.036 0.91 0.39 1.01 0.51 25.79 0.97 14.96 0.53
MD2 Comfort 1.65 0.094 1.18 0.40 1.60 0.40 24.84 1.83 15.31 0.21
Stress –0.23 0.023 0.88 0.35 0.58 0.20 24.69 1.44 14.94 0.39
Main effects
    Diet MS 1.17 0.41 1.01 0.30 1.61a 0.79 25.85 1.33 14.96 0.45
MD 1.00 0.36 1.03 0.39 1.10b 0.62 24.77 1.53 15.12 0.35
    Environment Comfort 1.63a 0.41 1.14 0.29 1.91a 0.52 25.38 1.77 15.13 0.37
Stress 0.04b 0.004 0.90 0.34 0.80b 0.42 25.24 1.28 14.95 0.43
    Diet NS3 NS 0.0319 NS NS
    Environment <0.0001 NS 0.0002 NS NS
    Interaction NS NS NS NS NS
a,bMean values within a row with unlike superscript letters were significantly different (P < 0.05).
1MS =methionine supplementation.
2MD = methionine deficient.
3NS = Not significant.

Heat stress significantly decreased the DWG (1.63 vs. 0.04 g; P < 0.0001) while MS had no effect on this variable.

No effect of the interaction between diet and temperature was observed on abdominal fat. Animals that received the MS diet exhibited higher levels of abdominal fat (1.61 vs. 1.10%; P = 0.0319). Regarding the environment, the percentage of abdominal fat was higher in animals that remained at the thermal comfort temperature (1.91 vs. 0.80%; P = 0.0002). The treatments (comfort MS, comfort MD, stress MS, and stress MD) did not influence the proportional weights of the liver, breast, and legs.

Reactive Oxygen Species Production, Oxidative Stress Markers, and Enzymatic Activity in the Liver

Hydrogen peroxide production was influenced by both of the study variables (Fig. 2). Increased H2O2 production was observed in the HS animals (0.0802 vs. 0.0692 nmol of ROS produced per minute per milligram of protein; P = 0.0042). Regarding diet, ROS production was increased in the MD diet animals (0.0808 vs. 0.0686 nmol of ROS produced per minute per milligram of protein; P = 0.0020).

Figure 2.
Figure 2.

Production of mitochondrial reactive oxygen species (ROS) in the liver of the quails exposed to thermal comfort or heat stress (HS) and fed with methionine supplementation (MS) or methionine deficient (MD) diet. The results are expressed in nanomoles of ROS produced per minute per milligram of protein. The results are shown as the average, and the standard deviation is represented by the vertical bars. The different letters between the treatment groups represent a significant difference (P < 0.05). Ptn = Protein.


Total GSH concentrations, TBARS levels, catalase, and GP-x activities in the liver are summarized in Table 3.

View Full Table | Close Full ViewTable 3.

Analysis of oxidative stress markers and antioxidant enzyme activities in the liver

GP-x3 activity
Cat4 activity
Diet - Environment Diet × Environment Mean SD Mean SD Mean SD Mean SD
MS5 Comfort 13.70 3.23 7.57b 2.15 172.46bc 8.08 29.91c 4.80
Stress 10.15 0.37 9.63b 1.78 245.72a 37.63 89.16b 12.11
MD6 Comfort 9.12 1.22 6.15b 1.12 162.54c 18.04 31.66c 3.33
Stress 8.63 2.28 13.14a 3.01 195.23b 14.91 203.92a 34.56
Main effects
    Diet MS 11.92a 2.86 9.10 1.62 209.09 36.23 59.54 7.82
MD 8.87b 1.75 10.14 2.67 178.88 23.24 117.79 23.08
    Environment Comfort 11.41a 3.33 7.36 3.85 167.49 14.28 30.79 4.04
Stress 9.39b 1.74 11.39 2.42 220.47 37.95 146.54 21.06
    Diet 0.0273 NS7 0.0037 <0.0001
    Environment 0.0018 <0.0001 <0.0001 <0.0001
    Interaction NS 0.0386 0.0392 <0.0001
a–cMean values within a row with unlike superscript letters were significantly different (P < 0.05).
1GSH = glutathione. Expressed as μg/mg of protein.
2TBARS = thiobarbituric acid reactive substances. Expressed as nmol TBARS/mg of protein.
3GP-x = glutathione peroxidase. Expressed as nmol of oxidized nicotinamide adenine dinucleotide phosphate /mg of protein per minute.
4Cat = catalase. Expressed as units/mg of protein.
5MS = methionine supplementation.
6MD = methionine deficient.
7NS= Not significant.

The total GSH concentration was influenced by both MS and HS. Higher GSH levels were observed in animals that received the MS diet (11.92 vs. 8.87 μg/mg of protein; P = 0.0273) and in animals kept in thermal comfort (11.41 vs. 9.39 μg/mg of protein; P = 0.0018).

Lipid peroxidation was evaluated by analyzing TBARS in the liver. The HS quails had increased TBARS levels in comparison to birds kept in thermal comfort (11.39 vs. 7.36 nmol TBARS/mg of protein; P < 0.0001). It was also possible to observe an interaction of the factors on TBARS levels (P = 0.0386), with the highest values found in animals that were subjected to HS and the MD diet (13.14 nmol TBARS/mg of protein; P = 0.0386).

Glutathione peroxidase activity in the quail livers were influenced by the interaction between temperature and diet (P = 0.0392). The enzymatic activity levels were higher in HS animals fed with MS diet (245.72 nmol of oxidized NADPH per mg of protein per minute).

Catalase activity was also influenced by an interaction between the 2 factors (P < 0.0001). The highest levels of catalase activity were found in birds subjected to HS and the MD diet (203.92 U/mg of protein).

We also analyzed the uric acid and creatinine plasma concentrations as well as the CK activity (Table 4). Uric acid concentrations were influenced by the interaction between diet and environment and the highest concentration was observed in HS birds fed with MS diet (P = 0.008).

View Full Table | Close Full ViewTable 4.

Plasma analysis of uric acid, creatinine, and creatine kinase (CK) activity levels

Uric acid, mg/dL
Creatinine, mg/dL
CK activity, units/L
Diet - Environment Diet × Environment Mean SD Mean SD Mean SD
MS1 Comfort 6.13b 1.41 0.20c 0.0002 2,707.83a 138.75
Stress 10.52a 2.26 0.23b 0.0052 2,377.00a 176.99
MD2 Comfort 6.43b 1.93 0.20c 0.0003 2,068.33a 150.40
Stress 5.42b 0.70 0.30a 0.002 1,620.33b 203.68
Main effects
    Diet MS 8.32 2.91 0.20 0.0001 2,388.10 176.11
MD 5.93 1.48 0.27 0.049 1,989.70 131.44
    Environment Comfort 6.28 1.62 0.22 0.04 2,542.04 183.18
Stress 7.97 3.10 0.25 0.05 1,844.30 173.05
    Diet 0.0023 <0.0001 NS3
    Environment 0.0237 0.0049 0.0347
    Interaction 0.0008 0.0049 0.0442
a–cMean values within a row with unlike superscript letters were significantly different (P < 0.05).
1MS = methionine supplementation.
2MD = methionine deficient.
3NS= Not significant.

Similarly, creatinine concentration (P = 0.0049) and CK activity levels (P = 0.0442) were also influenced by interactions between the factors. The highest creatinine levels (0.30 mg/dL) and lowest CK activity values (1,620.33 U/L) were observed in animals that were on the MD diet and exposed to HS.


When birds are subjected to HS, environmental and postural mechanisms such as feed intake reduction are used primarily in an attempt to reduce metabolic heat production and increase heat dissipation (Mujahid et al., 2005). In our study, HS led to a significant decrease in feed intake, concomitant with an increase in water consumption. Daily weight gain was decreased in birds exposed to HS. Contrary to our expectations, MS, which has been linked directly to better performance in birds (Kauomar et al., 2011), had no effect on DWG. This result can be attributed to the age of the birds used in the present study, as the birds were not at the stage of greater muscle deposition. The age of the birds may have also influenced fat deposition in animals that were fed the MS diet. According to Nikolova et al. (2007), abdominal fat deposition in broiler chickens increased from 0.73% at 35 d old to 1.06% at 49 d old.

The exposure of birds to high temperatures has been associated constantly with decreased production efficiency. Additionally, heat-induced acute (Lin et al., 2006) and chronic stress (Yang et al., 2010) have been linked to metabolic changes that are related to oxidative stress. According to the authors, high temperature–induced stress caused increased ROS production and decreased activity of the mitochondrial respiratory chain.

Reactive oxygen species are produced mainly as a function of proton leakage during phosphorylative oxidation; however, the mechanism of ROS production in heat stressed birds is not yet fully known (Tan et al., 2010). Possible mechanisms of ROS production under HS conditions might be linked to protein oxidation and decreased activity in respiratory chain complexes or to other forms of mitochondrial damage. Such damage could be associated with body temperature (due to increased ROS production) followed by decreased body weight gain in birds that exhibit higher body temperatures (Mujahid et al., 2005). Because chemical and biochemical reaction rates increase with temperature, it is likely that an increase in body temperature would lead to ROS generation via the accelerated metabolic reactions in the cells and tissues (Lin et al., 2006).

Contrary to our expectations, H2O2 production was influenced not only by ambient temperature but also by MS. A higher rate of H2O2 production was observed in HS quails and in those fed a diet without MS.

Uncoupling proteins have been described as elements that are capable of decreasing ROS production, as they contribute to the depolarization of the internal mitochondrial membrane (Sack, 2006). Studies have shown that UCP-encoding mRNA expression may vary with age (Gasparino et al., 2012) and environmental conditions (Del Vesco and Gasparino, 2012), among other factors. The same studies suggest that decreased expression levels of UCP-encoding mRNA are associated with increased ROS production; in 28 d old quails, the expression of UCP-encoding mRNA is decreased in comparison to 7-d-old birds, and the expression was also decreased in meat quails that were exposed to acute HS.

Cellular mechanisms such as UCP, which reduce ROS production, cannot fully eliminate this process that occurs constantly during energy generation, in which 2 to 4% of the total amount of O2 used as an electron acceptor is not fully reduced to water (Bottje et al., 2006). Therefore, to maintain the redox state and prevent oxidative stress, cells need to rely on mechanisms that are capable of eliminating the free radicals being produced, such as antioxidant enzymes.

In the present study, birds were subjected to HS to evaluate whether this factor could induce increased ROS production. Methionine supplementation in the diet was studied mainly to evaluate the possible role of this AA in antioxidant mechanisms, which might thus confer protection against cell damage from oxidative stress.

Antioxidant enzymes such as catalase and GP-x are essential in the elimination of ROS. Both are able to protect cells mainly against H2O2. Studies have shown that under conditions where ROS production is elevated, such as in animals subjected to HS, the activities of these enzymes also increase (Tan et al., 2010; Yang et al., 2010).

The results of our study are in agreement with data available in the literature. Catalase activity is increased in birds exposed to HS in comparison to birds kept exclusively at thermal comfort. Catalase activity was highest in the HS birds subjected to a diet without MS. This result could be related to a cellular compensation mechanism to quench increased H2O2 production because other defense mechanisms, such as GP-x activity, were decreased in birds that received the MD diet.

In addition to GSH, the GSH system comprises the enzymes glutathione oxidase, GP-x, and glutathione reductase; therefore, the antioxidant activity is dependent on the activity of the entire system. In our study, the greatest GP-x activity was found in HS birds and fed a MS diet. This result can be explained by the cellular need for increased GP-x activity while under stress conditions (Yang et al., 2010) and by the larger amount of GSH available while under MS that enable the GSH antioxidant system to operate adequately.

Glutathione is a tripeptide synthetized from glutamate, glycine, and cysteine. Cysteine can be synthetized from homocysteine from the precursor methionine (Shoveller et al., 2005); as such, when dietary methionine is available in an adequate amount, larger quantities can be directed towards cysteine synthesis via the transsulfuration pathway.

Some studies have shown that under stress conditions, the majority of metabolically available homocysteine is directed towards GSH synthesis. This might be explained either by the increased activity of cystathionine β-synthase, which is the enzyme responsible for cysteine production (Persa et al., 2004), or by the decreased activity of methionine synthase, which is the enzyme responsible for the synthesis of methionine from a homocysteine substrate; decreased activity of methionine synthase results from the oxidation of the cobalamine domains, which temporarily inactivates the enzyme (Muratore, 2010).

We observed higher amounts of GSH in animals that were fed the MS diet; this result could be explained by the increased amount of available methionine that led to an increased capacity to synthesize total GSH. Likewise, we also found lower GSH amounts in HS animals; we believe that this result is due to a greater requirement for GSH to target the increased ROS production under stress conditions. Previously, Mager and Kruijff (1995) observed that cells exposed to high temperatures presented with a decreased maintenance of GSH levels.

In addition to taking part in GSH synthesis, methionine can act as a direct antioxidant. In cells, several ROS can react with methionine residues to generate methionine sulfoxide. This product can be catalyzed back to methionine by the enzyme methionine sulfoxide reductase, in a thioredoxin-dependent reaction. Therefore, methionine residues can act as direct antioxidants and protect the proteins where these residues are located and several other molecules from ROS (reviewed by Luo and Levine, 2009).

Uric acid, like methionine, has been reported in the literature as one of many elements that exhibits antioxidant activity. At the physiological pH range, it is commonly found as urate, a powerful ROS scavenger released into the bloodstream by deleterious reactions such as hemoglobin auto-oxidation or peroxide production by macrophages. Urate can inactivate an oxidant before they can react with biological molecules such as DNA, proteins, and lipid membranes (Sautin and Johnson, 2008).

Birds possess specific mechanisms that contribute to increased urate concentrations in the blood, such as the absence of the enzyme uricase and the ability to encapsulate uric acid with proteins. Studies have indicated a relationship between higher uric acid concentrations and the decreased presence of oxidative stress markers (Klandorf et al., 2001; Simoyi et al., 2002).

In our work, we observed increased uric acid concentrations in birds that were exposed to HS and a MS diet. This suggests that stress demands an increased concentration of this antioxidant in the plasma and that this increased level results from MS because the presence of this AA supplement can increase feed intake and concomitantly glycine intake, which is a necessary element for uric acid synthesis. Methionine supplementation (Bunchasak et al., 2006) and increased feed intake have been linked to increased plasma uric acid concentration levels in broiler chickens (Machin et al., 2004).

Thiobarbituric acid reactive substances in the liver were used as biological markers of the extent of lipid peroxidation damage caused by increased H2O2 production. Creatine kinase activity in quail plasma was also used as a marker, as increased concentrations of this enzyme in the plasma have been linked to decreased membrane integrity and were observed in birds exposed to HS (Willemsen et al., 2011). However, CK contains thiol groups that are easily oxidized by ROS, rendering it relatively unstable because the formation of internal disulfide bonds causes a loss of activity (Gunst et al., 1998).

We observed an interaction between the effects of ambient temperature and MS on the levels of TBARS and CK activity; increased TBARS and decreased CK activity were found in HS animals that were fed a MD diet. Animals exposed to HS but fed the MS diet retained their TBARS levels and the enzymatic activities of these animals were significantly similar to those kept in thermal comfort.

In agreement with our study, Mujahid et al. (2009) observed increased TBARS levels in birds exposed to HS. The authors suggested that the negative effects caused by high temperatures could be attenuated with a dietary supplement of olive oil, which was linked to possible increases in UCP-encoding mRNA expression and decreases in ROS production.

Previous studies in the literature also link oxidative stress and decreased CK activity (Glaser et al., 2010; Aksenova et al., 2002), possibly via thiol group oxidation. The activity of the enzyme can be preserved by endogenous GSH, which serves as a protective agent during half-life of the enzyme in circulation; the loss of activity under certain conditions cannot be recovered when the extracellular GSH concentration is decreased, even in the presence of thiol-reducing agents (Gunst et al., 1998).

In light of the known effects on bird performance that are caused by HS and MS, we evaluated the creatinine concentrations in quail plasma to establish which condition induced greater muscle catabolism. We found that the plasma creatinine concentrations were highest in animals that were fed the MD diet and subjected to HS.

These results allow us to suggest that under HS conditions, in which the H2O2 production was highest, MS could attenuate ROS-induced damage, possibly consequent to the increased antioxidant activity of GSH, GPx activity, and uric acid concentration, as these were present in larger amounts in animals that were exposed to HS and received MS feed.




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