Effects of free and dipeptide forms of methionine supplementation on oxidative metabolism of broilers under high temperature.

Our previous studies have shown that methionine supplementation could help to attenuate the effects of heat stress on the metabolism of broiler chickens. Here we investigated for the first time the effects of methionine supplementation in the form of DL-methionyl-DL-methionine on broilers subjected to heat stress during the growth phase. Broilers were divided into two groups; one group was reared under thermoneutral conditions and the other under continuous heat stress (30 ±â€¯1 °C, 60% relative humidity). Both groups were subdivided into three dietary treatments: a methionine-deficient (MD) diet, a diet supplemented with free methionine (DL-M), and a diet supplemented with methionine dipeptide (DL-MM). Broilers raised under chronic heat stress had lower feed intake and weight gain than broilers raised under thermoneutral conditions (P < 0.05). There were no differences in animal performance between methionine-supplemented diets (DL-M and DL-MM). Heat-stressed birds had significantly higher heterophil/lymphocyte (H/L) ratio than thermoneutral birds. Under heat stress, broilers fed DL-M and DL-MM diets had lower H/L ratio than birds fed the MD diet. Higher concentrations of carbonylated proteins and lower concentration of reduced glutathione were observed in broilers raised under heat stress. In comparing heat-stressed broilers, we found that birds fed the DL-M diet had lower concentrations of thiobarbituric acid-reactive substances and carbonylated proteins than those fed the MD diet (P < 0.05). Higher expression of glutathione peroxidase (GPX) and glutathione synthetase (GSS) genes was observed in heat-stressed broilers (P < 0.05). Under heat stress, the MD diet increased GPX expression compared with other diets. Under thermoneutral conditions, the DL-M diet resulted in the highest GSS expression. There was a negative correlation between DNA methylation and GPX and GSS expression. Our results showed that supplementation of broiler diets with free methionine or methionine dipeptide may help attenuate the effects of heat stress through enhanced activation of genes related to the glutathione antioxidant system. Methionine effects were found for gene regulation, gene expression, and post-translational processing.

Lippia gracilis Schauer essential oil as a growth promoter for Japanese quail.

The use of antibiotics as performance enhancers in animal feeding is declining, so Lippia gracilis Schauer essential oil (LGSEO) could be used as a potential substitute for the conventionally used growth promoters. The LGSEO contains components such as carvacrol and thymol, which kill and/or control pathogenic bacteria, increase population of beneficial organisms, act against oxidative processes and onto nutrient digestibility and absorption. The aim of this study was to investigate the action and the effects of LGSEO as a growth promoter in the diet of Japanese quail by examining their productive performance, intestinal microbiology, blood biochemical parameters, hepatic thiobarbituric acid reactive substances (TBARS) content and intestinal gene expression. A total of 252 two-day-old quail (Coturnix coturnix japonica) were assigned to 3 treatments in 7 replicates, using 12 birds per experimental unit. The treatments consisted of a basal diet, basal diet + LGSEO at 400 mg/kg of diet and basal diet + chemical antimicrobial (bacitracin methylene disalicylate) at 500 mg/kg of diet. The experimental period was 34 days. The highest feed intake (P < 0.01) was found in the group receiving the conventional antimicrobial, whereas the best feed conversion (P < 0.01) was shown by the animals receiving LGSEO. Escherichia coli growth was restricted in the quail receiving the growth promoters. Salmonella spp. growth was controlled by the treatment containing the conventional antimicrobial. There was no difference between the treatments (P > 0.05) for the concentration of aspartate aminotransferase and alanine aminotransferase enzymes in the blood or hepatic TBARS content. Birds receiving negative-control treatment exhibited a higher expression of sodium-glucose cotransporter (SGLT1), while those receiving the treatment with essential oil showed lower catalase (CAT) and glutathione peroxidase (GPX7) expressions compared to the conventional antimicrobial and control groups, respectively. Lippia gracilis Schauer essential oil is a powerful performance enhancer for Japanese quail by virtue of its abilities to improve their intestinal environment, balance the microbial population and reduce energy expenditure for oxidative processes.

Effects of methionine hydroxy analogue supplementation on the expression of antioxidant-related genes of acute heat stress-exposed broilers.

We evaluated the effects of heat stress (HS) and methionine supplementation on biological markers of stress and expression of the genes for superoxide dismutase (SOD), thioredoxin (TRx), thioredoxin reductase 1 (TRxR1) and methionine sulfoxide reductase A (MsrA) in broilers aged 1 to 21 or 22 to 42 days. The broilers were divided into two treatments, one with the recommended level of methionine supplementation (MS, supplementation of dl-2-hydroxy-4-methylthiobutanoic acid (dl-HMTBA)) and one without methionine supplementation (MD). The animals were maintained at a temperature of thermal comfort or one of HS (38°C for 24 h). Mortality was only observed in 42-day-old broilers exposed to HS and fed the MD diet, and the rate was 5%. Starter period: we observed an interaction effect between diet and temperature on the gene expression of TRxR1 and MsrA, and expression of these genes was higher in the HS animals that received the MS diet than that in birds with the MD diet. Grower period: the expression of SOD, TRxR1 and MsrA genes, activities of aspartate aminotransferase (AST) and creatine kinase (CK) and content of creatinine were influenced by both study variables. In the HS animals, the expression of these genes, AST activity and creatinine content increased and CK activity decreased. In the animals on the MD diet, the expression of these genes and AST and creatinine values were higher and the CK activity was lower than those for the birds on the MS diet. Our results indicated that under HS conditions, the supplementation with dl-HMTBA could mitigate major damage caused by stress through the action on some genes related to TRx complex activity.

Age-related oxidative stress and antioxidant capacity in heat-stressed broilers.

We aimed to evaluate the effects of acute heat stress (HS) and age on the redox state in broilers aged 21 and 42 days. We evaluated the expression of genes related to antioxidant capacity, the production of hydrogen peroxide (H2O2), and the activity of antioxidant enzymes in the liver, as well as oxidative stress markers in the liver and plasma. The experiment had a completely randomized factorial design with two thermal environments (thermoneutral and HS, 38°C for 24 h) and two ages (21 and 42 days). Twenty-one-day-old animals exposed to HS showed the highest thioredoxin reductase 1 (TrxR1) (P<0.0001) and glutathione synthetase (GSS) (P<0.0001) gene expression levels. Age influenced the expression of the thioredoxin (Trx) (P=0.0090), superoxide dismutase (SOD) (P=0.0194), glutathione reductase (GSR) (P<0.0001) and glutathione peroxidase 7 (GPx7) (P<0.0001) genes; we observed greater expression in birds at 21 days than at 42 days. Forty-two-day-old HS birds showed the highest H2O2 production (222.31 pmol dichlorofluorescein produced/min×mg mitochondrial protein). We also verified the effects of age and environment on the liver content of Glutathione (GSH) (P<0.0001 and P=0.0039, respectively) and catalase (CAT) enzyme activity (P=0.0007 and P=0.0004, respectively). Higher GSH content and lower CAT activity were observed in animals from the thermoneutral environment compared with the HS environment and in animals at 21 days compared with 42 days. Broilers at 42 days of age had higher plasma creatinine content (0.05 v. 0.01 mg/dl) and higher aspartate aminotransferase activity (546.50 v. 230.67 U/l) than chickens at 21 days of age. Our results suggest that under HS conditions, in which there is higher H2O2 production, 21-day-old broilers have greater antioxidant capacity than 42-day-old animals.

Effects of selenium supplementation on the oxidative state of acute heat stress-exposed quails.

This study aimed to evaluate the effect of heat stress (HS) and selenium supplementation on markers of stress, meat quality and gene expression. For this, meat quails of 42 days of age were fed a diet that either met [0.33 mg/kg, nutritional demand for selenium (SS)] or did not meet [0.11 mg/kg, selenium deficient (SD)] the nutritional demands for selenium during the 7 days of evaluation. In addition, the animals were kept at either a thermal comfort temperature (25 °C) or exposed to HS (38 °C for 24 h). Glutathione synthetase (GSS), glutathione reductase (GSR) and uncoupling protein (UCP) gene expression were influenced by the interaction between temperature and diet. Animals subjected to HS and fed the SS diet exhibited the highest GSS and GSR gene expression. In terms of UCP gene expression, the lowest values were observed in HS animals on the SD diet. Glutathione peroxidase 7 (GPX7) gene expression, body temperature (BT) and creatine kinase (CK) activity were influenced by both selenium supplementation and HS. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) activity and creatinine content all were influenced by the diet/environment interaction. The highest AST activity, ALT activity and creatinine levels were observed in animals that were both on the SD diet and exposed to HS. HS animals also exhibited an increased heterophil/lymphocyte ratio and lower triiodothyronine (T3) hormone levels than birds that remained at the comfortable temperature. Animals subjected to HS and fed with selenium supplemented diet showed better results regarding gene expression and, thus, better results for the activities of enzymes used as stress markers, which could be due to the higher antioxidant capacity provided by the action of the studied genes.

Mitochondrial gene expression in the liver and muscle of high and low feed efficiency Japanese quail layers subjected to different environmental temperatures.

We evaluated the adenine nucleotide translocator (ANT) and cytochrome oxidase subunit III (COX III) mRNA expressions in the muscle and liver of Japanese quails presenting high and low feed efficiency (FE), and subjected them to three different environmental temperatures: comfort, heat stress (38°C), and cold stress (10°C). ANT mRNA expression was lower in the liver of heat-stressed animals. In the muscle, higher ANT and COX III mRNA expressions were observed in high-FE and cold-stressed animals. In the liver, much higher expression of COX III mRNA was observed in cold-stressed animals. These results suggest a possible correlation between the genes involved in energy production by the mitochondria and FE phenotypes, and that environmental temperature can affect the ANT and COX III mRNA expressions. Japanese quails presenting different FE levels respond differently to environmental stimuli.

The effect of heat stress on GHR, IGF-I, ANT, UCP and COXIII mRNA expression in the liver and muscle of high and low feed efficiency female quail.

1. A study was conducted to test the hypothesis that feed efficiency (FE) correlated with the expression of genes from the somatotropic axis and mitochondrial genes involved in energy production, and that the environment to which the birds are exposed influenced the expression of such genes. 2. Quails were divided into High-FE and low-FE groups and maintained in a comfortable or heat stress (HS) (38°C for 24 h) environment to evaluate changes in insulin-like growth factor I (IGF-I), growth hormone receptor (GHR), adenine nucleotide translocator (ANT), uncoupling protein (UCP) and cytochrome oxidase subunit III (COX III) messenger ribonucleic acid (mRNA) expression in liver and muscle tissues. 3. High-FE quails (0.28 g/g) presented a higher final body weight, greater weight gain and a better feed conversion ratio than low-FE birds (0.18 g/g). High-FE birds showed greater IGF-I mRNA expression in the liver and muscle and greater GHR mRNA expression in the muscle. 4. Environmental effects only affected GHR expression in the liver, with quails under comfortable conditions exhibiting greater GHR expression than quails subjected to HS. 5. There was a significant interaction between FE and environmental temperature on ANT mRNA expression in the liver. The greatest ANT mRNA expression was observed for high FE-birds that remained under comfortable conditions. 6. In the liver, UCP mRNA expression did not differ among the quails and was not affected by environment or efficiency. However, comparisons of the low- and high-FE birds revealed higher levels of UCP mRNA in the muscle of low-FE birds. 7. COX III mRNA expression in the liver was dependent on environmental temperature and FE. Higher COX III mRNA expression was observed in animals that remained under comfortable conditions, and high-FE birds exhibited higher expression levels compared to low-FE birds. 8. These results suggest a correlation between IGF-I, GHR, ANT, UCP and COX III gene expression and FE and that environmental temperature could affect the expression of some of these genes.

Acute heat stress and dietary methionine effects on IGF-I, GHR, and UCP mRNA expression in liver and muscle of quails.

This study evaluated the expression of insulin-like growth factor I (IGF-I), growth hormone receptor (GHR), and uncoupling protein (UCP) mRNA in muscle and liver of quails that were in thermal comfort or exposed to heat stress and that were fed diets with or without methionine supplementation. Meat quails were fed a diet that either met the nutritional demands for methionine (MS) or did not meet this demand (methionine-deficient diet, MD). The animals were either kept at a thermal comfort temperature (25°C) or exposed to heat stress (38°C for 24 h starting on the 6th day). RNA was extracted from liver and breast muscle, and cDNA was synthesized and amplified using quantitative reverse transcription-polymerase chain reaction. Animals that were fed the MS diet and remained at the thermal comfort temperature exhibited increased IGF-I mRNA expression in the liver (0.56 AU). The GHR mRNA expression in the liver and muscle was influenced by both the study variables. Animals receiving the MS diet showed higher GHR expression, while increased expression was observed in animals at the thermal comfort temperature. The UCP mRNA expression in the muscle was influenced by both methionine supplementation and heat stress. Higher expression was observed in animals that received the MD diet (2.29 vs 3.77 AU) and in animals kept in thermal comfort. Our results suggest that heat stress negatively affects the expression of growth-related genes and that methionine supplementation is necessary to appropriately maintain the levels of IGF-I, GHR, and UCP transcripts for animal metabolism.

Effects of methionine supplementation on the redox state of acute heat stress-exposed quails.

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.

Expression of calpastatin and myostatin genes associated with lamb meat quality.

Calpastatin (CAST) is an endogenous calpain inhibitor and its main function is to modulate the proteolytic action of enzymes responsible for post-mortem myofibril deterioration. The myostatin gene (GDF-8) acts as a negative regulator of skeletal muscle growth. The expression of these two genes, as well as their interaction, affects the quality of the meat, especially the tenderness phenotype. We evaluated the genetic groups Santa Inês, ½ Dorper-Santa Inês and ½ White Dorper-Santa Inês, slaughtered with 2.0 mm, 2.5 mm and 3.0 mm of fat thickness, comparing the levels of expression of the CAST and GDF-8 genes with the weight performance and carcass traits, especially the shear force values. We found significantly higher expression of myostatin and calpastatin in the Santa Inês genetic group. The ½ Dorper-Santa Inês genetic group had the lowest expression of these genes when slaughtered with 2.0 and 2.5 mm of fat thickness. In conclusion, the Santa Inês breed had the lowest phenotype values for meat tenderness, and the ½ Dorper-Santa Inês breed had the best performance for this characteristic. We suggest that high levels of the expression of the CAST and GDF-8 genes are associated with lower values of lamb meat tenderness, and that tenderness is related to the stage of muscular growth and development.

Production of reactive oxygen species, gene expression, and enzymatic activity in quail subjected to acute heat stress.

The aim of the present study was to evaluate the effect of acute heat stress on the production of mitochondrial reactive oxygen species (ROS), the gene expression of the avian uncoupling protein (avUCP) and glutathione peroxidase (GPX 7), and the activity of the enzyme GPX in the liver of meat quail. Two groups of 15 meat quail (Coturnix coturnix japonica) that were 23 d of age were initially housed individually in metallic cages. A period of 7 d was provided for the 2 bird groups to adapt to the cages and to a thermoneutral environment at 25°C with 60% relative humidity. At 30 d of age, 15 quail were exposed to a heat stress (HS) treatment of 34°C for 24 h, humidity 60%, whereas control quail (n = 15) were kept at 25°C. To analyze the production of ROS, 4 quail from each treatment group were slaughtered, and their livers were collected for mitochondrial isolation and to measure the subsequent production of ROS by the mitochondria. Additionally, the livers of 6 animals from each treatment group were collected for total RNA extraction. The cDNA was amplified using primers specific for the target genes, and expression was analyzed using the real-time PCR reaction (qRT-PCR). Five animals from each treatment group were slaughtered to analyze glutathione peroxidase (GPx) activity, which was determined by using of hydrogen peroxide (H2O2), and based on measuring the amount nicotinamide adenine dinucleotide phosphate oxidized. A greater amount of mitochondrial ROS was found in HS animals (0.34 vs. 0.22 nm of ROS produced min(-1) · mg(-1) of protein, P < 0.05) for the reactions that contained only rotenone and in the reactions that were performed with rotenone and antimycin (0.31 vs. 0.23 nm of ROS produced min(-1) · mg(-1) of protein, P < 0.05). Concomitantly, the birds that were subjected to acute heat stress and had a greater amount of ROS production expressed less avUCP mRNA [0.75 arbitrary units (AU) vs. 0.87 AU, P < 0.05] and more GPX 7 mRNA (2.37 AU vs. 1.17 AU, P < 0.01). The HS quail displayed significantly greater GPx activity in their hepatocytes (47.8 vs. 39.6 nmol of NADPH oxidized per mg of protein per minute, P < 0.05). Thus, acute heat stress at 34°C for 24 h affects the production of mitochondrial ROS, the expression of avUCP and GPX 7 mRNA, and the activity of the GPx enzyme in the liver of meat quail.

Dietary methionine effects on IGF-I and GHR mRNA expression in broilers.

This study aimed to evaluate liver and breast muscle insulin-like growth factor I (IGF-I) and growth hormone receptor (GHR) gene expression between broilers fed different methionine levels and sources. Broiler chicks were 22 to 42 days old, distributed in 5 treatments (control diet, DL1 - 0.08% DL-methionine, DL2 - 0.24% DL-methionine, MHA-FA1 - 0.11% methionine hydroxy analogue-free acid, and MHA-FA2 - 0.33% methionine hydroxy analogue-free acid). The broilers were euthanized by cervical dislocation. RNA was extracted from liver and breast muscle, followed by cDNA synthesis and amplification using qRT-PCR. DL2 methionine supplementation provided best animal performance results. GHR and IGF-I gene expression in the muscle tissue was not affected by methionine supplementation. IGF-I gene expression in the liver was higher in animals fed methionine supplementation than in animals fed control diet. IGF-I mRNA levels in broilers fed DL2 were greater than DL1 (1.56 vs 0.97 AU) and greater than MAH-FA1 and MAH-FA2. Broilers fed DL2 increased significantly GHR gene expression in the liver than animals fed the control diet. Addition of methionine improved animal performance by stimulating synthesis and release of growth factor.

Expression of growth genes in response to glycerol use in Japanese quail diets.

Glycerol can be used as a substitute for corn for feeding poultry, but there are concerns about how it may affect growth performance and health of the birds. We evaluated the expression of mRNA of growth hormone (GH) and insulin-like growth factor I (IGF-I) in 35-day-old Japanese quails fed different glycerol levels (0, 4, and 8% dietary glycerol instead of corn). Total RNA was extracted from the breast muscle and cDNA was amplified with the use of specific primers for these genes using real-time PCR. Quails fed the diet with 8% glycerol supplementation had significantly lower GH mRNA and IGF-I mRNA expression than those fed no glycerol or 4% glycerol. No significant effect of the treatments was found on quail weight gain or feed intake. Feed conversion ratio was influenced by dietary glycerol levels: the group fed 8% glycerol displayed the worst feed conversion ratio (2.54) compared with that of quail fed the control diet (2.35) or 4% glycerol (2.36). Considering quail performance and the expression of the genes GH and IGF-I, a level of 4% glycerol can be used in quail feeding without any harmful effects.

Age-related changes in mitochondrial UCP, ANT and COX III gene expression in the breast muscle of quails (Coturnix coturnix japonica).

Efficient conversion of food into body mass has been associated with altered gene expression of some proteins of the electron transport chain. We evaluated the effect of age on mRNA expression of uncoupling protein, adenine nucleotide translocase and cytochrome c oxidase complex III in quails. One-day-old broiler quails were sacrificed after 7, 14, 21, or 28 days. Six quails of each age were slaughtered by cervical dislocation. Total RNA was extracted from the breast muscle and reverse transcribed into cDNA. Real-time PCR analysis was performed using specific primers for the genes. As the quails aged, there was reduced expression of all three genes. The greatest reduction when comparing 7- and 28-day-old birds was observed in the uncoupling protein mRNA levels (67%), followed by adenine nucleotide translocase (56%) and cytochrome c oxidase complex III (30%). We conclude that expression of some mitochondrial genes of the electron transport chain changes with age in quails.