Effects of Methionine Supplementation on the Expression of Protein Deposition-Related Genes in Acute Heat Stress-Exposed Broilers

The objective of this study was to evaluate the effect of heat stress and methionine supplementation on the gene expression of insulin-like growth factor I (IGF-I), growth hormone receptor (GHR), phosphatidylinositol 3-kinase, and regulatory 1 (PI3KR1) in the liver, as well as the expression of the atrogin 1 and cathepsin L2 (CTSL2) genes in the breast muscle of broilers. Broilers from 1–21 and 22–42 days of age were divided into three treatments related to methionine supplementation as follows: without methionine supplementation (MD), recommended level of methionine (DL1), and excess supplementation of methionine (DL2). The animals were either maintained at a thermal comfort temperature or exposed to heat stress (HS) (38°C for 24 hours, starting on day 20 or day 41 for experiments 1 and 2, respectively). The heat stress increased the body temperature at both ages. Starter period: The HS animals presented increased plasma creatinine content (P<0.0001) and the highest CTSL2 gene expression (P<0.0001). The methionine supplementation increased the IGF-I (P = 0.0144) and GHR (P = 0.0011) gene expression and decreased the CTSL2 (P = 0.0004) and atrogin 1 (P = 0.0012) gene expression. Grower period: Significant effects for the interaction between supplementation and environment were observed for GHR (P = 0.0252) and CTSL2 (P = 0.0011) gene expression. The highest GHR expression was observed in animals that remained in thermal comfort on the DL2 diet, and the lowest expression occurred in the HS animals fed the MD diet. For CTSL2, the HS animals fed the MD diet presented the highest CTSL2 gene expression, and the lowest expression was observed in the animals maintained at thermal comfort on DL1 and DL2 diets. Only methionine supplementation had effect on atrogin-1 gene expression (P<0.0001), with higher methionine content in the diet lower atrogin-1 gene expression was observed. Our results suggest that heat stress induces greater protein degradation and that methionine supplementation could induce protein deposition because methionine increased the expression of genes related to protein synthesis and decreased the expression of genes related to protein breakdown.     

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 ± 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.     

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.     

Prolonged selenium deficient diet in MsrA knockout mice causes enhanced oxidative modification to proteins and affects the levels of antioxidant enzymes in a tissue-specific manner

The methionine sulfoxide reductase (Msr) system (comprised of MsrA and MsrB) is responsible for reducing methionine sulfoxide (MetO) to methionine. One major form of MsrB is a selenoprotein. Following prolonged selenium deficient diet (SD), through F2 generation, the MsrA ? / ?mice exhibited higher protein–MetO and carbonyl levels relative to their wild-type (WT) control in most organs. More specifically, the SD diet caused alteration in the expression and/or activities of certain antioxidants as follows: lowering the specific activity of MsrB in the MsrA ? / ?cerebellum in comparison to WT mice; lowering the activities of glutathione peroxidase (Gpx) and thioredoxin reductase (Trr) especially in brains of MsrA ? / ?mice; elevation of the cellular levels of selenoprotein P (SelP) in most tissues of the MsrA ? / ?relative to WT. Unexpectedly, the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) were mainly elevated in lungs and hearts of MsrA ? / ?mice. Moreover, the body weight of the MsrA ? / ?mice lagged behind the WT mice body weight up to 120 days of the SD diet. In summary, it is suggested that the lack of the MsrA gene in conjunction with prolonged SD diet causes decreased antioxidant capability and enhanced protein oxidation.     

Effects of gingerols-rich extract of ginger on growth performance,serum metabolites,meat quality and antioxidant activity of heat-stressed broilers

In order to investigate the effects of dietary ginger extract (GE) enriched in gingerols on broilers under heat stress (HS) from 21 to 42 days of age, a total of 144 Ross 308 male broilers were randomly allocated to three groups with six replicates of eight broilers per replicate. Broilers in the control group were raised at 22 °C and fed a basal diet, and broilers in the other two groups were raised under cyclic HS (34 °C from 9:00 to 17:00 and at 22 °C for the rest of the time) and fed the basal diet with or without 1000 mg/kg GE. Supplementation of GE improved (P < 0.05) final body weight, average daily gain and feed conversion ratio of broilers under HS, and tended (P < 0.1) to increase breast muscle yield. The alterations of serum total protein, albumin, total cholesterol levels and aspartate aminotransferase activity under HS were reversed (P < 0.05) by GE, which also decreased (P < 0.05) serum triglyceride level and alanine aminotransferase activity. The decreased redness (a* value) and increased drip loss of breast muscle induced by HS were restored (P < 0.05) by GE. Moreover, GE supplementation increased (P < 0.05) total antioxidant capacity and decreased (P < 0.05) malondialdehyde content in liver and breast muscle, and increased (P < 0.05) glutathione peroxidase activity in serum and breast muscle. In conclusion, dietary GE supplementation restored growth performance, serum metabolites and meat quality of broilers under HS possibly by improving antioxidant activity.     

Protective effects of taurine against oxidative stress in the heart of MsrA knockout mice

Taurine has been shown to have potent anti‐oxidant properties under various pathophysiological conditions. We reported previously a cellular dysfunction and mitochondrial damage in cardiac myocytes of methionine sulfoxide reductase A (MsrA) gene knockout mice (MsrA^?/?). In the present study, we have explored the protective effects of taurine against oxidative stress in the heart of MsrA^?/? mice with or without taurine treatment. Cardiac cell contractility and Ca²⁺ dynamics were measured using cell‐based assays and in vivo cardiac function was monitored using high‐resolution echocardiography in the tested animals. Our data have shown that MsrA^?/? mice exhibited a progressive cardiac dysfunction with a significant decrease of ejection fraction (EF) and fraction shortening (FS) at age of 8 months compared to the wild type controls at the same age. However, the dysfunction was corrected in MsrA^?/? mice treated with taurine supplement in the diet for 5 months. We further investigated the cellular mechanism underlying the protective effect of taurine in the heart. Our data indicated that cardiac myocytes from MsrA^?/? mice treated with taurine exhibited an improved cell contraction and could tolerate oxidative stress better. Furthermore, taurine treatment reduced significantly the protein oxidation levels in mitochondria of MsrA^?/? hearts, suggesting an anti‐oxidant effect of taurine in cardiac mitochondria. Our study demonstrates that long‐term treatment of taurine as a diet supplement is beneficial to a heart that is vulnerable to environmental oxidative stresses. J. Cell. Biochem. 113: 3559–3566, 2012. © 2012 Wiley Periodicals, Inc.     

Effects of different levels of selenium on growth performance and immunocompetence of broilers under heat stress

An experiment was undertaken to evaluate the effect of dietary selenium (Se) levels on growth performance and immune competence of broilers under heat stress. Birds were raised in either a thermoneutral (TN, 23.9°C constant) or heat stress conditions (HS, 23.9°C to 38°C cycling) and were fed a corn-soybean meal basal diet supplemented with Se at 0, 0.2 and 0.4 mg/kg. A total of 240 one-day-old male broiler chicks were randomly assigned to six groups; each group had four replicates of 10 birds. Body weight and feed intake were not influenced by dietary Se, while feed conversion was significantly improved by a Se-supplementation of 0.2 mg/kg. HS significantly reduced body weight, feed intake and feed conversion. Numbers of abdominal exudate cells (AEC), percentage of macrophages in AEC, phagocytic macrophages, internalized opsonised and unopsonised sheep red blood cells (SRBC) were significantly increased by dietary Se. Both primary and secondary antibody responses were characterised by increasing titres of antibody to SRBC by dietary Se when birds were exposed to HS (p < 0.05). Lymphoid organ weights, antibody responses, incidence of macrophages in AEC, and phagocytic ability of macrophages were also significantly reduced under HS. These results indicated that HS severely reduced growth performance and immunocompetence of broilers, whereas the immune response of broilers improved by dietary Se supplementation under HS.     

Effect of epigallocatechin gallate on growth performance and antioxidant capacity in heat-stressed broilers

This study investigated the effects of epigallocatechin gallate (EGCG) on the growth performance and antioxidant capacity of 35-d-old broilers exposed to heat stress. Broilers, 14 d of age, were divided into four groups with six replicates per group (eight chickens/replicate). Thermoneutral group (Group TN) was fed the basal diet and maintained at 28°C for 24 h/d. The heat-stressed groups were housed at 35°C for 12 h/d and 28°C for 12 h/d and fed the basal diet supplemented with EGCG at 0, 300 and 600 mg/kg diet (Groups HS0, HS 300 and HS600, respectively). Compared with Group TN, heat-stressed groups showed significantly reduced gain, feed intake and serum total protein and glucose levels; inhibited serum alkaline phosphatase activities; and increased serum levels of uric acid, cholesterol and triglycerides and the activity of serum creatine kinase, lactate dehydrogenase and aspartate aminotransferase (p < 0.05). Compared with Group HS0, Group HS600 exhibited an increased gain and feed intake; and normalised blood parameters and enzyme activities. Compared with Group TN, the expression of antioxidant-related liver proteins was decreased in Group HS0 and increased in Groups HS300 and HS600 (p < 0.05). The results suggest that EGCG can improve the growth performance and alleviate the oxidant damage by modulating the antioxidant properties of broilers.     

Differential expression of small heat shock proteins in the brain of broiler embryo; the effects of embryonic thermal manipulation

Broilers are more vulnerable to high temperatures than mammals due to the feather cover, lack of sweat glands, fast growth and intensive breeding in commercial systems. Thermal stresses affect the function of various organs and change the expression profiles of hundreds of genes in the different tissues of broilers. Thermal manipulation (TM) during embryogenesis can increase heat tolerance in growing broilers. Small heat shock proteins (SHSPs) are a group of HSPs which participate in many cellular functions like response to different stressors. However, their role in the thermotolerance has not been fully elucidated. Ninety fertilized eggs were randomly divided into three groups (30 eggs/group; 10 eggs/replicate). Normal control (NC) eggs were incubated at 37.5 °C throughout the incubation period whereas heat stress (HS) and cold stress (CS) groups were kept at 41 °C and 33 °C from 15 to 17th day of incubation for 3 h each day, respectively. On day 20, samples from the cerebrums were harvested for histopathology and mRNA expression analyses of HSPB1, HSPB5, HSPB8, and HSPB9. There were no significant differences in survivability, defected embryos, hatchability, and body weight among treatments. TM had no major deleterious effects on the cerebral tissue except for mild degeneration in the HS group. HSPB1, HSPB5, HSPB8, and HSPB9 were expressed in the presence and absence of TM. All SHSP genes tested were downregulated in response to TM except for HSPB9 which was upregulated in the HS group. The highest change in gene expression due to TM observed for HSPB1. This study presents a broader understanding of mechanisms underlying response to TM in broilers. The results suggest that HSPB1, HSPB5, HSPB8, and HSPB9 are involved in thermotolerance in broilers and SHSPs could be involved in the gene expression profiling of TM. It may propose the use of nutritional supplements in the poultry industry to modulate SHSPs.     

Flash dietary methionine supply over growth requirements in pigs: Multi-facetted effects on skeletal muscle metabolism

Dietary methionine affects protein metabolism, lean gain and growth performance and acts in the control of oxidative stress. When supplied in large excess relative to growth requirements in diets for pigs, positive effects on pork quality traits have been recently reported. This study aimed to decipher the molecular and biochemical mechanisms affected by a dietary methionine supply above growth requirements in the loin muscle of finishing pigs. During the last 14 days before slaughter, crossbred female pigs (n = 15 pigs/diet) were fed a diet supplemented with hydroxy-methionine (Met5; 1.1% of methionine) or not (CONT, 0.22% of methionine). Blood was sampled at slaughter to assess key metabolites. At the same time, free amino acid concentrations and expression or activity levels of genes involved in protein or energy metabolism were measured in the longissimus lumborum muscle (LM). The Met5 pigs exhibited a greater activity of creatine kinase in plasma when compared with CONT pigs. The concentrations of free methionine, alpha-aminobutyric acid, anserine, 3-methyl-histidine, lysine, and proline were greater in the LM of Met5 pigs than in CONT pigs. Expression levels of genes involved in protein synthesis, protein breakdown or autophagy were only scarcely affected by the diet. Among ubiquitin ligases, MURF1, a gene known to target creatine kinase and muscle contractile proteins, and OTUD1 coding for a deubiquitinase protease, were up-regulated in the LM of Met5 pigs. A lower activity of citrate synthase, a reduced expression level of ME1 acting in lipogenesis but a higher expression of PPARD regulating energy metabolism, were also observed in the LM of Met5 pigs compared with CONT pigs. Principal component analysis revealed that expression levels of many studied genes involved in protein and energy metabolism were correlated with meat quality traits across dietary treatments, suggesting that subtle modifications in expression of those genes had cumulative effects on the regulation of processes leading to the muscle transformation into meat. In conclusion, dietary methionine supplementation beyond nutritional requirements in pigs during the last days before slaughter modified the free amino acid profile in muscle and its redox capacities, and slightly affected molecular pathways related to protein breakdown and energy metabolism. These modifications were associated with benefits on pork quality traits.     

Prolonged selenium-deficient diet in MsrA knockout mice causes enhanced oxidative modification to proteins and affects the levels of antioxidant enzymes in a tissue-specific manner

The methionine sulfoxide reductase (Msr) system (comprised of MsrA and MsrB) is responsible for reducing methionine sulfoxide (MetO) to methionine. One major form of MsrB is a selenoprotein. Following prolonged selenium deficient diet (SD), through F2 generation, the MsrA - / -mice exhibited higher protein-MetO and carbonyl levels relative to their wild-type (WT) control in most organs. More specifically, the SD diet caused alteration in the expression and/or activities of certain antioxidants as follows: lowering the specific activity of MsrB in the MsrA - / -cerebellum in comparison to WT mice; lowering the activities of glutathione peroxidase (Gpx) and thioredoxin reductase (Trr) especially in brains of MsrA - / -mice; elevation of the cellular levels of selenoprotein P (SelP) in most tissues of the MsrA - / -relative to WT. Unexpectedly, the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) were mainly elevated in lungs and hearts of MsrA - / -mice. Moreover, the body weight of the MsrA - / -mice lagged behind the WT mice body weight up to 120 days of the SD diet. In summary, it is suggested that the lack of the MsrA gene in conjunction with prolonged SD diet causes decreased antioxidant capability and enhanced protein oxidation.     

Hypolipidemic effect of the edible mushroom Agaricus blazei in rats subjected to a hypercholesterolemic diet

The effects of Agaricus blazei intake on the lipid profile of animals fed a hypercholesterolemic diet were evaluated. Thirty-two female Fisher rats were divided into four groups and given the standard AIN-93 M diet (C), this diet?+?1 % A. blazei (CAb), a hypercholesterolemic diet with 25 % soybean oil and 1 % cholesterol (H) or this diet?+?1 % A. blazei (HAb) for 6 weeks. Food intake, weight gain, liver and serum lipid profiles, activity of aminotransferases [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)], and creatinine and urea levels as well as abdominal fat weight were measured. Histological analysis of kidney and liver tissue was also performed. The HAb group had a higher food intake, but a lower weight gain as compared to group H. This resulted in a significant decrease in abdominal fat weight, to values close to those of groups C and CAb. Supplementing the hypercholesterolemic diet with A. blazei promoted a significant reduction in total and non-HDL cholesterol, as well as in the atherogenic index, as compared to group H, and this effect was more pronounced in the serum. There was no hepatotoxic effect caused by the supplementation of the diets with the mushroom. We conclude that in our experimental model and in the concentration used, A. blazei was effective in improving the lipid profile of the animals.     

Effects of Arginine and Methionine on the Growth Depression of Rats Fed Diets High in Glycine

In order to determine if the growth retardation by dietary exceess glycine could be prevented by the addition of arginine and/or methionine, weanling rats were fed a 25% casein diet (standard) or a 10% casein diet (low protein diet) with a supplement of several combinations of glycine, arginine, or methionine.

The changes in body weight, urinary creatinine, and kidney transamidinase activity were determined. The growth depression effect by excess glycine was prevented considerably in animals receiving standard diet and completely in animals receiving low protein diet by the addition of arginine and methionine to the high glycine diets.

The total urinary creatinine was increased by the supplement of both glycine and arginine, while the growth rate was not invariably raised and kidney transamidinase activity had a tendency to decrease.     

Effect of dietary lysine on performance and expression of electron transport chain genes in the pectoralis major muscle of broilers

The aim of this study was to evaluate the effect of dietary lysine on performance, protein deposition and respiratory chain gene expression in male broilers. A total of 252 Cobb 500 broilers were distributed, in a completely randomized design, into four treatments with seven replicates of nine birds per experimental unit. Experimental treatments consisted of diets based on corn and soybean meal, with four levels of digestible lysine: 1.016%, 1.099%, 1.182% and 1.265%. The increase in the level of digestible lysine in the diet provided higher weight gains, feed efficiency and body protein deposition. Birds fed the lowest level of dietary lysine (1.016%) showed a lower expression of genes such as NADH dehydrogenase subunit I (ND1), cytochrome b (CYTB) and cytochrome c oxidase subunits I (COX I), II (COX II) and III (COX III), displaying the worst performance and body protein deposition. This demonstrates the relationship existing between the expression of the evaluated genes and the performance responses. In conclusion, results indicate that broilers fed diets with higher levels of digestible lysine have increased messenger RNA expression of some genes coded in the mitochondrial electron transport chain (ND1, CYTB, COX I, COX II and COX III). It may be stated that diets with proper levels of digestible lysine, within the ‘ideal protein’ concept, promote the expression of genes, which increases the mitochondrial energy, thereby fostering body protein deposition and the performance of broilers in the starter phase.     

Effect of chronic cyclic heat stress on the intestinal morphology,oxidative status and cecal bacterial communities in broilers

The objective of this study was to examine the effects of chronic cyclic heat stress (HS) on the intestinal morphology, oxidative stress and cecal bacterial communities of broilers. One-day-old Arbor Acres (AA) male broilers (n = 100) were acclimated for 3 weeks and then randomly allocated into two groups, normal control (NC) group (22 ± 1 °C, 24 h/day) and HS group (32 ± 1 °C, 10 h/day lasted for 2 weeks). At 35 d of age, intestinal segments (duodenum, jejunum and ileum) and cecal digesta were collected for detection. HS affected intestinal morphology, inducing epithelial cell abscission, inflammatory cell infiltration, and lamina propria edema. Compared with the NC group, HS significantly decreased (P < 0.01) villus height (VH) and the VH-to-crypt depth (CD) ratio (VCR), increased (P < 0.05) CD in the duodenum and ileum, but had no effect on the VH in the jejunum. Moreover, HS induced oxidative stress with antioxidant enzymes activity decreasing (P < 0.05) while malondialdehyde (MDA) content increasing (P < 0.05) in small intestine. Pearson's correlation analysis indicated that MDA content was negatively correlated with VH (P < 0.05). The result of 16S rRNA sequencing showed that HS exposure impacted cecal microbiota alpha diversity (phylogenetic diversity whole-tree index) and beta diversity. Based on principal coordinate analysis (PCoA) plots for weighted UniFrac metrics and unweighted pair group method with arithmetic mean (UPGMA), there were 8 discriminative features at the genus level (linear discriminant analysis score > 2). Parabacteroides, Saccharimonas, Romboutsia and Weissella were reduced, while Anaerofustis, Pseudonocardia, Rikenella and Tyzzerella were enriched in heat-stressed broilers. Collectively, these results indicated that chronic cyclic HS induced oxidative stress that caused damage to intestinal villus-crypt structures, and then altered the cecal microflora profile.