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.

High leucine levels affecting valine and isoleucine recommendations in low-protein diets for broiler chickens.

Four experiments were conducted to estimate the optimal standardized ileal digestible (SID) level of branched-chain amino acids in low-protein diets during the starter, grower, and finisher periods, using the response surface methodology, and to study their effects on performance and mRNA expression of genes involved in the mechanistic target of rapamycin (mTOR) pathway of broiler chickens from 8 to 21 D of age. In experiments 1, 2, and 3, a total of 1,500 Cobb male broiler chickens were assigned to 15 diets of a central composite rotatable design (CCD) of response surface methodology containing 5 levels of SID Leu, Val, and Ile with 5 replicate pens of 20 birds each. A 3-factor, 5-level CCD platform was used to fit the second-order polynomial equation of broiler performance. In experiment 4, a total of 540 8-day-old Cobb male broiler chickens were distributed in a completely randomized 2 x 3 x 3 factorial arrangement with 2 SID Leu levels (1.28 or 1.83%), 3 SID Val levels (0.65, 0.90, or 1.20%), and 3 SID Ile levels (0.54, 0.79, or 1.09%) for a total of 18 treatments with 5 replicate cages of 6 birds each. High Leu levels impaired (P < 0.05) gain:feed when birds were fed marginal Val or Ile diets. However, gain:feed was restored when both Val and Ile were supplemented to reach adequate or high levels. High Leu levels increased (P < 0.05) mRNA expression of S6K1 and eEF2 genes only in birds fed high Ile levels. Dietary SID Leu, Val, and Ile levels required for gain:feed optimization in low-protein diets were estimated at 1.37, 0.94, and 0.87% during the starter period; 1.23, 0.82, and 0.75% during the grower period; and 1.15, 0.77, and 0.70% during the finisher phase, respectively. Higher Val and Ile levels are required to optimize the effect of Leu supplementation on mRNA expression of mTOR pathway genes in the pectoralis major muscle of broilers from day 1 to 21 after hatch.

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.

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.