Dietary supplementation with canthaxanthin and 25-hydroxycholecalciferol on the incubation performance and fertility of European quail breeders.

This study assessed the effects of combined supplementation with canthaxanthin (Cx) and 25-hydroxycholecalciferol (25-OH-D3) on incubation performance, fertility, and chick quality in European quail breeders. A total of 240 birds were distributed in a completely randomized design with 5 diets and 8 replicates. The animals were fed a basal diet containing 50 µg of vitamin D3 or the basal diet supplemented with 3 ppm Cx and 34.5 µg 25-OH-D3, 6 ppm Cx, and 69 µg 25-OH-D3, 9 ppm Cx and 103.5 µg 25-OH-D3, or 12 ppm Cx and 138 µg 25-OH-D3. Incubation performance was analyzed in 2 periods (32 and 38 wk). Breeders aged 32 wk produced eggs with higher hatchability (P = 0.024), hatchability of fertile eggs (P = 0.026) and lower initial plus mid embryonic mortality (P = 0.021), whereas 38-week-old breeders generated chicks with a higher length at hatching (P < 0.001) and lower final plus pipped embryonic mortality (P = 0.021). In both age groups, Cx + 25-OH-D3 levels had a quadratic effect on egg fertility (P < 0.001), hatchability of total (P < 0.001), and fertile eggs (P < 0.001). The fertility and the number of sperm cells in the perivitelline membrane was analyzed in two periods (26 and 40 wk). A quadratic effect of diet and days after mating on both parameters (P < 0.05) was observed. Eggs from supplementing breeders showed a high fertility (P < 0.001) and sperm cell counts (P < 0.001) for up to 7 and 3 d after mating, respectively, then the control group. Moreover, the supplementation of quail breeder diets with 6 ppm Cx + 69 µg 25-OH-D3 enhances sperm cell longevity in sperm storage tubules, hatchability of total and fertile eggs, fertility, and chick quality, especially in older quail's breeders and reduces embryonic mortality.

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.

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.

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.

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.

Effect of heat stress and feeding phosphorus levels on pig electron transport chain gene expression.

The purpose of this study was to evaluate the effect of temperature and different levels of available phosphorus (aP) on the expression of nine genes encoding electron transport chain proteins in the Longissimus dorsi (LD) muscle of pigs. Two trials were carried out using 48 high-lean growth pigs from two different growth phases: from 15 to 30 kg (phase 1) and from 30 to 60 kg (phase 2). Pigs from growth phase 1 were fed with three different levels of dietary aP (0.107%, 0.321% or 0.535%) and submitted either to a thermoneutral (24°C and RH at 76%) or to a heat stress (34°C and RH at 70%) environment. Pigs from growth phase 2 were fed with three different levels of dietary aP (0.116%, 0.306% or 0.496%) and submitted either to a thermoneutral (22ºC and RH at 77%) or to a heat stress (32ºC and RH at 73%) environment. Heat stress decreased (P<0.001) average daily feed intake at both growth phases. At 24°C, pigs in phase 1 fed the 0.321% aP diet had greater average daily gain and feed conversion (P<0.05) than those fed the 0.107% or 0.535% while, at 34°C pigs fed the 0.535% aP had the best performance (P<0.05). Pigs from phase 2 fed the 0.306% aP had best performance in both thermal environments. Gene expression profile was analyzed by quantitative real-time polymerase chain reaction. Irrespective of growing phase, the expression of six genes was lower (P<0.05) at high temperature than at thermoneutrality. The lower expression of these genes under high temperatures evidences the effects of heat stress by decreasing oxidative metabolism, through adaptive physiological mechanisms in order to reduce heat production. In pigs from phase 1, six genes were differentially expressed across aP levels (P<0.05) in the thermoneutral and one gene in the heat stress. In pigs from phase 2, two genes were differentially expressed across aP levels (P<0.05) in both thermal environments. These data revealed strong evidence that phosphorus and thermal environments are key factors to regulate oxidative phosphorylation with direct implications on animal performance.

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.

The effect of glycerol on mRNA expression of growth hormone, insulin-like growth factor, and mitochondrial breast muscle genes of Japanese quail.

1. A study was carried out to evaluate the expression of growth hormone (GH), insulin-like growth factor I (IGF-I), mitochondrial adenine nucleotide translocase (ANT), cytochrome oxidase III (COX III) and avian uncoupling protein (avUCP) genes in 14-d-old Japanese quail that were fed different levels of glycerol (0%, 4%, and 12% dietary glycerol) which replaced maize as an energy source. 2. Total RNA was extracted from the breast muscle, and cDNA was amplified using real-time PCR with primers specific to the examined genes. 3. Quail fed the diet with 12% glycerol supplementation presented higher growth hormone (GH) mRNA expression than did those fed 0% glycerol. Supplementation with 12% glycerol negatively influenced IGF-I mRNA expression and reduced ANT mRNA expression in comparison with the treatment with no glycerol. COX III mRNA expression in the pectoralis superficialis muscle was decreased by 26% in quail fed 12% glycerol compared with those fed 0 and 4% glycerol. 4. There was no difference in UCP mRNA expression between quail fed 0 and 4% glycerol; however, UCP expression was reduced (73%) in birds fed the 12% glycerol diet compared with the level in those fed the 4% glycerol diet. 5. The inclusion of 4% glycerol in the diet produced results similar to those of the diet with no glycerol. Based on quail performance and the expression of the GH, IGF-I, ANT, COX III and UCP genes, 4% glycerol can be used in quail feeding without any harmful effects.