Ontogeny of hepatic metabolism in two broiler lines divergently selected for the ultimate pH of the Pectoralis major muscle.

BACKGROUND: Nutrient availability during early stages of development (embryogenesis and the first week post-hatch) can have long-term effects on physiological functions and bird metabolism. The embryo develops in a closed structure and depends entirely on the nutrients and energy available in the egg. The aim of this study was to describe the ontogeny of pathways governing hepatic metabolism that mediates many physiological functions in the pHu + and pHu- chicken lines, which are divergently selected for the ultimate pH of meat, a proxy for muscle glycogen stores, and which differ in the nutrient content and composition of eggs. RESULTS: We identified eight clusters of genes showing a common pattern of expression between embryonic day 12 (E12) and day 8 (D8) post-hatch. These clusters were not representative of a specific metabolic pathway or function. On E12 and E14, the majority of genes differentially expressed between the pHu + and pHu- lines were overexpressed in the pHu + line. Conversely, the majority of genes differentially expressed from E18 were overexpressed in the pHu- line. During the metabolic shift at E18, there was a decrease in the expression of genes linked to several metabolic functions (e.g. protein synthesis, autophagy and mitochondrial activity). At hatching (D0), there were two distinct groups of pHu + chicks based on hierarchical clustering; these groups also differed in liver weight and serum parameters (e.g. triglyceride content and creatine kinase activity). At D0 and D8, there was a sex effect for several metabolic pathways. Metabolism appeared to be more active and oriented towards protein synthesis (RPS6) and fatty acid ß-oxidation (ACAA2, ACOX1) in males than in females. In comparison, the genes overexpressed in females were related to carbohydrate metabolism (SLC2A1, SLC2A12, FoxO1, PHKA2, PHKB, PRKAB2 and GYS2). CONCLUSIONS: Our study provides the first detailed description of the evolution of different hepatic metabolic pathways during the early development of embryos and post-hatching chicks. We found a metabolic orientation for the pHu + line towards proteolysis, glycogen degradation, ATP synthesis and autophagy, likely in response to a higher energy requirement compared with pHu- embryos. The metabolic orientations specific to the pHu + and pHu- lines are established very early, probably in relation with their different genetic background and available nutrients.

Effects of sex and fasting/refeeding on hepatic AMPK signaling in chickens (Gallus gallus).

The alpha-1 isoform of chicken AMPK situates on the Z-chromosome, in contrast, the other isoforms in birds and the mammalian AMPKα1 are located on the autosomes. The present study aimed to investigate the role of hepatic AMPK signaling in adaptation to nutritional status and the potential sex-specific response in chickens. Hepatic genes and proteins were compared between the two sexes immediately after hatching. From 20d of age, chicks from each sex received feed treatments: Control was fed ad libitum; Fasted was starved for 24 h; Refed was fed for 4 h after a 24 h fasting. As a result, hepatic AMPKα1 mRNA level in males was significantly higher at both ages compared to females, due to the presence of Z-chromosomes. However, this did not make this kinase "male-bias" as it was eventually compensated at a translational level, which was not reported in previous studies. The protein levels and activation of AMPKα were even lower in newly-hatched male compared to female chicks, accompanied with a higher FAS and SREBP-1 gene expressions. Accordingly, hepatic G6PC2 mRNA levels in males were significantly lower associated with lower plasma glucose levels after hatching. Fasting activated hepatic AMPK, which in turn inhibited gene expression of GS, FAS and SREBP-1, and stimulated the downstream G6PC2 in both sexes. These changes recovered after refeeding. In conclusion, AMPK plays a role in adaptation to nutritional environment for both sexes. The Z-linked AMPK did not exert a sex-specific signaling, due to a "translational compensation" of AMPKα1.

Methionine and cysteine deficiencies altered proliferation rate and time-course differentiation of porcine preadipose cells.

Methionine (Met) is an essential sulfur amino acid (AA) limiting growth and is the precursor of cysteine (Cys), the rate-limiting factor in the synthesis of glutathione, and the main intracellular non-enzymatic antioxidant. This study aimed at determining the effects of limited supplies in Met and(or) Cys in early aspects of adipose tissue development and oxidative stress in differentiated adipocytes. Incremental reductions in Met (70, 40, and 0 µM) were compared with Met 100 µM (control dose) in porcine preadipocytes cultured in media without or with Cys (250 µM). In Cys-deprived media, both the absence (0 µM) and the lowest dose of Met (40 µM) reduced preadipocyte proliferation. Adding Cys in media only partly compensated for this decrease. On the opposite, mild Met deficiency (70 µM) did not alter preadipocyte proliferation in media without or with Cys. Strong Met deficiency (40 µM) also reduced differentiation and lipid accumulation into preadipose cells. Mild Met deficiency also reduced preadipocyte differentiation when Cys was present in the culture media, whereas in Cys-deprived media, percent of differentiated cell was similar and intracellular lipid content was slightly higher at Met 70 µM than at Met 100 µM. Finally, incremental reductions in Met in media with or without Cys lowered reactive oxygen species (ROS) production by differentiated cells. These results demonstrate the strong dependency of porcine adipogenesis to sulfur AA supplies. Strong Met deficiency decreases both proliferation and differentiation, whereas mild deficiency only alters differentiation.

Thermal manipulation of the chicken embryo triggers differential gene expression in response to a later heat challenge.

BACKGROUND: Meat type chickens have limited capacities to cope with high environmental temperatures, this sometimes leading to mortality on farms and subsequent economic losses. A strategy to alleviate this problem is to enhance adaptive capacities to face heat exposure using thermal manipulation (TM) during embryogenesis. This strategy was shown to improve thermotolerance during their life span. The aim of this study was to determine the effects of TM (39.5 °C, 12 h/24 vs 37.8 °C from d7 to d16 of embryogenesis) and of a subsequent heat challenge (32 °C for 5 h) applied on d34 on gene expression in the Pectoralis major muscle (PM). A chicken gene expression microarray (8 × 60 K) was used to compare muscle gene expression profiles of Control (C characterized by relatively high body temperatures, Tb) and TM chickens (characterized by a relatively low Tb) reared at 21 °C and at 32 °C (CHC and TMHC, respectively) in a dye-swap design with four comparisons and 8 broilers per treatment. Real-time quantitative PCR (RT-qPCR) was subsequently performed to validate differential expression in each comparison. Gene ontology, clustering and network building strategies were then used to identify pathways affected by TM and heat challenge. RESULTS: Among the genes differentially expressed (DE) in the PM (1.5 % of total probes), 28 were found to be differentially expressed between C and TM, 128 between CHC and C, and 759 between TMHC and TM. No DE gene was found between TMHC and CHC broilers. The majority of DE genes analyzed by RT-qPCR were validated. In the TM/C comparison, DE genes were involved in energy metabolism and mitochondrial function, cell proliferation, vascularization and muscle growth; when comparing heat-exposed chickens to their own controls, TM broilers developed more specific pathways than C, especially involving genes related to metabolism, stress response, vascularization, anti-apoptotic and epigenetic processes. CONCLUSIONS: This study improved the understanding of the long-term effects of TM on PM muscle. TM broilers displaying low Tb may have lower metabolic intensity in the muscle, resulting in decreased metabolic heat production, whereas modifications in vascularization may enhance heat loss. These specific changes could in part explain the better adaptation of TM broilers to heat.

Central role of 5'-AMP-activated protein kinase in chicken sperm functions.

Avian gametes present specific features related to their internal long-term mode of fertilization. Among other central actors of energetic metabolism control, it has been suspected that 5'-AMP-activated protein kinase (AMPK) influences sperm functions and thus plays a key role in fertilization success. In the present work, we studied AMPK localization and function in chicken sperm incubated in vitro. Effects of the pharmacological AMPK activators (AICAR, metformin) and the AMPK inhibitor compound C were assessed by evaluating AMPKalpha (Thr(172)) phosphorylation (by Western blotting), semen quality (by viability, motility, and ability to perform acrosome reaction), and energetic metabolism indicators (lactate, ATP). Localization of AMPK in subcellular sperm compartments was evaluated by immunocytochemistry. Total AMPK was found in all compartments except for the nucleus, but the phosphorylated form phospho-Thr(172)-AMPK was essentially localized in the flagellum and acrosome. AMPK activators significantly improved AMPK phosphorylation, sperm motility (increased by 40% motile, 90% progressive, and 60% rapid sperm), acrosome reaction and lactate production (increased by 40%) and viability. The AMPK inhibitor significantly reduced AMPK phosphorylation and percentages of motility (decrease by 25%), progressive energy (decrease by 35%), and rapid sperm (decreased by 30%), acrosome reaction, lactate production, and ATP release. The two activators differed in their effect on ATP concentration: AICAR stimulated ATP formation, whereas metformin did not. Our results indicate that AMPK plays a key role in the regulation of chicken sperm functions and metabolism. This action differs from that suggested in mammals, mainly by its crucial involvement in the acrosome reaction process.

Modulation of the insulin anabolic signalling cascade in growing chickens by n-3 PUFA.

n-3 PUFA are crucial for health and development. Their effects as regulators of lipid and glucose metabolism are well documented. They also appear to affect protein metabolism, especially by acting on insulin sensitivity. The aim of the present study was to investigate the role of n-3 PUFA, i.e. the precursor α-linolenic acid (ALA) 18:3n-3 or long-chain PUFA (LC-PUFA), in chickens, by focusing on their potential function as co-regulators of the insulin anabolic signalling cascade. Ross male broilers were divided into six dietary treatment groups. Diets were isoproteic (22 % crude protein) and isoenergetic (12·54 MJ metabolisable energy/kg) and contained similar lipid levels (6 %) provided by different proportions of various lipid sources: oleic sunflower oil rich in 18:1n-9 as control; fish oil rich in LC-PUFA; rapeseed and linseed oils providing ALA. The provision of diets enriched with n-3 PUFA, i.e. rich in LC-PUFA or in the precursor ALA, for 3 weeks improved the growth performance of chickens, whereas that of only the ALA diet enhanced the development of the pectoralis major muscle. At 23 d of age, we studied the insulin sensitivity of the pectoralis major muscle and liver of chickens after an intravenous injection of insulin or saline. The present results indicate that the activation patterns of n-3 PUFA are different in the liver and muscles. An ALA-enriched diet may improve insulin sensitivity in muscles, with greater activation of the insulin-induced 70 kDa ribosomal protein S6 kinase/ribosomal protein S6 pathway involved in the translation of mRNA into proteins, thereby potentially increasing muscle protein synthesis and growth. Our findings provide a basis on which to optimise dietary fatty acid provision in growing animals.

Short-term nutritional strategies before slaughter are effective in modulating the final pH and color of broiler breast meat.

The poultry meat industry is faced with various quality issues related to variations in the ultimate pH of breast meat. The aim of this study was to evaluate the possibility to control breast ultimate pH by distributing finishing diets varying in amino acid (AA) and energy content for a short period before slaughter. Experimental diets were distributed to PM3 broilers on the last 3 d before slaughter (36 d of age). They consisted of a control (C) diet (3,150 kcal/kg; 200 g/kg of CP; 10.0 g/kg of true digestible Lys) with adequate amounts of AA other than Lys, 6 diets isocaloric to the control diet including 3 Lys-deficient (8.0 g/kg) diets with an adequate (Lys-/AA), low (Lys-/AA-), or high (Lys-/AA+) amount of other essential AA calculated in relation to Lys, and 3 Lys-rich (12.0 g/kg) diets with an adequate (Lys+/AA), low (Lys+/AA-), or high (Lys+/AA+) amount of other essential AA calculated in relation to Lys, and 2 diets isoproteic to C with a high (3,300 kcal/kg, E+) or low (3,000 kcal/kg, E-) energy content. Broiler feed consumption and growth performance were slightly affected by AA and energy content during the finishing period. Feed intake (33-36 d) was lower with the Lys+/AA+ and E+, and FCR between 24 and 36 d was higher with the Lys-/AA- and E- than with the C diet. Body weight at d 36 was lower in Lys-/AA-, Lys+/AA+, and E+ than in C, whereas the breast meat yield and abdominal fatness were not affected by diet. Lower pH values were observed in broilers fed Lys-deficient diets containing a high amount of other AA (Lys-/AA+) than in broilers fed diets containing low (AA-) or adequate (AA) amounts of other AA. This study shows that it is possible to alter the pH of breast meat by changing AA profile over a short period before slaughter, with limited impact on broiler growth and carcass composition.

Implication of digestive functions and microbiota in the establishment of muscle glycogen differences between divergent lines for ultimate pH.

Both the quality of chicken meat and the quality of chicks are influenced by the level of breast muscle glycogen reserves. In order to study the role of digestive metabolism in establishing this muscular phenotype, we compared two divergent chicken lines for the ultimate pH (pHu) of the breast meat, a proxy for glycogen reserves. Males aged 4 weeks had twice the breast muscle glycogen content in the pHu- line (low pHu) than in the pHu + line (high pHu). The increase in glycogen reserves (pHu-) was associated with a higher relative weight of the proventriculus and gizzard, as well as better apparent ileal digestibility of nitrogen and calcium. The diversity of the cecal microbiota was comparable, but three bacterial genera (Lachnospira, Lachnospiraceae UCG-010, Caproiciproducens) varied between the lines. The differences observed could lead to down-regulation of carbon fixation in prokaryotes and of the citrate cycle in the pHu + line. RNA-seq analysis of the jejunum, the major site of nutrient absorption, revealed 149 genes differentially expressed (DE) between the lines, including several genes linked to immunity, hormonal response and circadian rhythms that are less expressed in pHu + animals. Others involved in cell migration and proliferation, and more generally tissue morphogenesis, also differed between the lines. Among the DE genes, several co-localized with Quantitative Trait Loci (QTL) controlling pHu and selection signatures identified in the divergent lines, such as the gene coding for ghrelin, a hormone regulating appetite.

The effect of albumen removal before incubation (embryonic protein under-nutrition) on the post-hatch performance, regulators of protein translation activation and proteolysis in neonatal broilers.

Albumen was removed from broiler eggs before the start of incubation to induce prenatal protein under-nutrition in chicken embryos. With this method, the direct effect of protein deficiency was investigated, differing from mammalian models manipulating the maternal diet where indirect, hormonal effects can interfere. Based on the estimated albumen/egg weight ratio, 10 % of albumen was removed with an 18G needle, after making a hole at the sharp end of the egg with another 18G needle. Eggs were taped thereafter. The sham group underwent the same procedure, except that no albumen was removed. Control eggs did not receive any treatment. The removal of albumen decreased both embryonic and post-hatch body weight up to day 7 compared with the control group. On embryonic day 18, embryos from the albumen-deprived group had higher plasma uric acid levels compared with the sham (P= 0·016) and control (P= 0·009) groups. Moreover, a lower plasma amino acid concentration was observed at hatch compared with the sham (P= 0·038) and control (P= 0·152) groups. These findings indicate an altered protein metabolism. At hatch, a higher mRNA expression of muscle ring finger-1 (MuRF1), a gene related to proteolysis, was observed in albumen-deprived chicks compared with the control and sham chicks, together with an up-regulated expression of atrogin-1 (another atrogene) at this time point in the male protein-deficient chicks. These findings suggest that muscle proteolysis is transiently increased by the removal of albumen before the start of incubation. No evidence was found for altered protein synthesis capacity and translational efficiency in albumen-deprived chicks.

Allantoic fluid metabolome reveals specific metabolic signatures in chicken lines different for their muscle glycogen content.

Nutrient availability in eggs can affect early metabolic orientation in birds. In chickens divergently selected on the Pectoralis major ultimate pH, a proxy for muscle glycogen stores, characterization of the yolk and amniotic fluid revealed a different nutritional environment. The present study aimed to assess indicators of embryo metabolism in pHu lines (pHu+ and pHu-) using allantoic fluids (compartment storing nitrogenous waste products and metabolites), collected at days 10, 14 and 17 of embryogenesis and characterized by 1H-NMR spectroscopy. Analysis of metabolic profiles revealed a significant stage effect, with an enrichment in metabolites at the end of incubation, and an increase in interindividual variability during development. OPLS-DA analysis discriminated the two lines. The allantoic fluid of pHu- was richer in carbohydrates, intermediates of purine metabolism and derivatives of tryptophan-histidine metabolism, while formate, branched-chain amino acids, Krebs cycle intermediates and metabolites from different catabolic pathways were more abundant in pHu+. In conclusion, the characterization of the main nutrient sources for embryos and now allantoic fluids provided an overview of the in ovo nutritional environment of pHu lines. Moreover, this study revealed the establishment, as early as day 10 of embryo development, of specific metabolic signatures in the allantoic fluid of pHu+ and pHu- lines.

Insulin immuno-neutralization in fed chickens: effects on liver and muscle transcriptome.

Chickens mimic an insulin-resistance state by exhibiting several peculiarities with regard to plasma glucose level and its control by insulin. To gain insight into the role of insulin in the control of chicken transcriptome, liver and leg muscle transcriptomes were compared in fed controls and "diabetic" chickens, at 5 h after insulin immuno-neutralization, using 20.7K-chicken oligo-microarrays. At a level of false discovery rate <0.01, 1,573 and 1,225 signals were significantly modified by insulin privation in liver and muscle, respectively. Microarray data agreed reasonably well with qRT-PCR and some protein level measurements. Differentially expressed mRNAs with human ID were classified using Biorag analysis and Ingenuity Pathway Analysis. Multiple metabolic pathways, structural proteins, transporters and proteins of intracellular trafficking, major signaling pathways, and elements of the transcriptional control machinery were largely represented in both tissues. At least 42 mRNAs have already been associated with diabetes, insulin resistance, obesity, energy expenditure, or identified as sensors of metabolism in mice or humans. The contribution of the pathways presently identified to chicken physiology (particularly those not yet related to insulin) needs to be evaluated in future studies. Other challenges include the characterization of "unknown" mRNAs and the identification of the steps or networks, which disturbed tissue transcriptome so extensively, quickly after the turning off of the insulin signal. In conclusion, pleiotropic effects of insulin in chickens are further evidenced; major pathways controlled by insulin in mammals have been conserved despite the presence of unique features of insulin signaling in chicken muscle.

Transcriptomic and metabolomic profiling of chicken adipose tissue in response to insulin neutralization and fasting.

BACKGROUND: Domestic broiler chickens rapidly accumulate adipose tissue due to intensive genetic selection for rapid growth and are naturally hyperglycemic and insulin resistant, making them an attractive addition to the suite of rodent models used for studies of obesity and type 2 diabetes in humans. Furthermore, chicken adipose tissue is considered as poorly sensitive to insulin and lipolysis is under glucagon control. Excessive fat accumulation is also an economic and environmental concern for the broiler industry due to the loss of feed efficiency and excessive nitrogen wasting, as well as a negative trait for consumers who are increasingly conscious of dietary fat intake. Understanding the control of avian adipose tissue metabolism would both enhance the utility of chicken as a model organism for human obesity and insulin resistance and highlight new approaches to reduce fat deposition in commercial chickens. RESULTS: We combined transcriptomics and metabolomics to characterize the response of chicken adipose tissue to two energy manipulations, fasting and insulin deprivation in the fed state. Sixteen to 17 day-old commercial broiler chickens (ISA915) were fed ad libitum, fasted for five hours, or fed but deprived of insulin by injections of anti-insulin serum. Pair-wise contrasts of expression data identified a total of 2016 genes that were differentially expressed after correction for multiple testing, with the vast majority of differences due to fasting (1780 genes). Gene Ontology and KEGG pathway analyses indicated that a short term fast impacted expression of genes in a broad selection of pathways related to metabolism, signaling and adipogenesis. The effects of insulin neutralization largely overlapped with the response to fasting, but with more modest effects on adipose tissue metabolism. Tissue metabolomics indicated unique effects of insulin on amino acid metabolism. CONCLUSIONS: Collectively, these data provide a foundation for further study into the molecular basis for adipose expansion in commercial poultry and identify potential pathways through which fat accretion may be attenuated in the future through genetic selection or management practices. They also highlight chicken as a useful model organism in which to study the dynamic relationship between food intake, metabolism, and adipose tissue biology.

Characterization of major elements of insulin signaling cascade in chicken adipose tissue: apparent insulin refractoriness.

The role of insulin in chicken adipose tissue appears weak or questionable. In a first study, proximal and distal components of the insulin signaling cascade were characterized in abdominal adipose tissue of fasted or fed chickens for the first time. Similar measurements were performed on epididymal adipose tissue from fasted or fed rats for comparison. Tyrosine phosphorylation of IR beta subunit, IRS-1 and Shc and phosphorylation of downstream components (Akt and MAPK ERK1/2) were significantly reduced as expected by fasting in rat, but not in chicken. Phosphorylation of MAPK P38 was increased by fasting in chicken but not in rat. Phosphorylation of AMPK was not affected in the conditions investigated in either species. Whatever the nutritional state, the protein levels of IR and IRS-1 were lower in chicken than in rat, whereas those of Shc, Akt, AMPK, MAPK ERK2 and MAPK P38 were similar in both species. In fed state, PI3K activity was higher in chicken than in rat. Insulin sensitivity of insulin cascade was further investigated in chicken adipose tissue following in vivo insulin neutralization for 1 or 5h in fed chickens. Insulin privation did not alter early insulin signaling steps (IRß, IRS-1 and Shc) or downstream elements (Akt, P70S6K, S6 ribosomal protein, AMPK, MAPK ERK2 and MAPK P38). Finally, phosphorylation of the transcription factor Creb was increased by 2-fold by 5h fasting or 5h insulin privation, most likely in response to an increase in plasma glucagon levels. Thus, insulin signaling is markedly different in chicken abdominal adipose tissue from that operating in mammals making chicken an interesting model of insulin resistance or refractoriness.

Research Note: Divergent selection for breast muscle ultimate pH affects egg quality traits in broiler breeders.

Two divergently selected broiler lines were created by selection for low (pHu-) or high (pHu+) Pectoralis major ultimate pH (pHu) in order to better understand the molecular mechanisms underlying meat quality traits in broilers and are also unique genetic resources reflecting low and high glycogen levels in chicken muscle. The present study aimed to reveal the correlated phenotypical changes of egg quality traits in broiler breeders from the 2 divergent lines at the 14th generation. Birds were reared on littered floor system until 18 wk of age and in individual cages up to 42 wk. Individual egg production was recorded daily from age at first egg to 42 wk. External (egg weight: EW and shape index: SI), internal (albumen height: AH, Haugh unit: HU, yolk index: YI, and yolk color: YC), and shell (shell percentage: ESP, thickness: EST and strength: ESS) characteristics of eggs in pHu- and pHu+ lines were measured in all eggs for 4 consecutive days at 26, 27, 28, 30, 31, 32, 41, and 42 wk of age. The pHu- line had significantly higher egg percentage than pHu+ (55.9 and 49.1%, respectively). The EW in pHu- line (57.2 g) was significantly lower than in pHu+ (59.0 g) and increased with age in both lines. The mean ESP, EST and ESS were lower in the pHu+ eggs compared to the pHu- line. ESP and EST decreased mainly from 26 to 27 wk of age and they had a stable trend with advancing age in the remaining weeks. AH and YI were lower in pHu- line eggs than in pHu+. YC was more intense and HU higher in pHu+ eggs than pHu- in pre-peak and peak laying period. In conclusion, these results showed that a divergent selection for muscle energy metabolism has led to correlated responses on internal and external egg quality traits and suggest that the production of good-quality eggs may be impaired in broiler breeders with low energy reserves.

A Divergent Selection on Breast Meat Ultimate pH, a Key Factor for Chicken Meat Quality, is Associated With Different Circulating Lipid Profiles.

Background: Chicken meat has become a major source of protein for human consumption. However, the quality of the meat is not yet under control, especially since pH values that are too low or too high are often observed. In an attempt to get a better understanding of the genetic and biochemical determinants of the ultimate pH, two genetic lines of broilers were divergently selected for low (pHu-) or high (pHu+) breast meat pHu. In this study, the serum lipidome of 17-day-old broilers from both lines was screened for pHu markers using liquid-chromatography coupled with mass spectrometry (LC-HRMS). Results: A total of 185 lipids belonging to 4 groups (glycerolipids, glycerophospholipids, sterols, sphingolipids) were identified in the sera of 268 broilers from the pHu lines by targeted lipidomics. The glycerolipids, which are involved in energy storage, were in higher concentration in the blood of pHu- birds. The glycerophospholipids (phosphatidylcholines, phosphatidylethanolamines) with long and polyunsaturated acyl chains were more abundant in pHu+ than in pHu- while the lysophosphatidylcholines and lysophosphatidylethanolamines, known to be associated with starch, were observed in higher quantity in the serum of the pHu- line. Finally, the concentration of the sterols and the ceramides, belonging to the sphingolipids class, were higher in the pHu+ and pHu-, respectively. Furthermore, orthogonal partial least-squares analyses highlighted a set of 68 lipids explaining 77% of the differences between the two broilers lines (R2Y = 0.77, Q2 = 0.67). Among these lipids, a subset of 40 predictors of the pHu value was identified with a Root Mean Squared Error of Estimation of 0.18 pH unit (R2Y = 0.69 and Q2 = 0.62). The predictive model of the pHu value was externally validated on 68 birds with a Root Mean Squared Error of Prediction of 0.25 pH unit. Conclusion: The sets of molecules identified will be useful for a better understanding of relationship between serum lipid profile and meat quality, and will contribute to define easily accessible pHu biomarkers on live birds that could be useful in genetic selection.

Nutrient sources differ in the fertilised eggs of two divergent broiler lines selected for meat ultimate pH.

The pHu+ and pHu- lines, which were selected based on the ultimate pH (pHu) of the breast muscle, represent a unique model to study the genetic and physiological controls of muscle energy store in relation with meat quality in chicken. Indeed, pHu+ and pHu- chicks show differences in protein and energy metabolism soon after hatching, associated with a different ability to use energy sources in the muscle. The present study aimed to assess the extent to which the nutritional environment of the embryo might contribute to the metabolic differences observed between the two lines at hatching. Just before incubation (E0), the egg yolk of pHu+ exhibited a higher lipid percentage compared to the pHu- line (32.9% vs. 27.7%). Although 1H-NMR spectroscopy showed clear changes in egg yolk composition between E0 and E10, there was no line effect. In contrast, 1H-NMR analysis performed on amniotic fluid at embryonic day 10 (E10) clearly discriminated the two lines. The amniotic fluid of pHu+ was richer in leucine, isoleucine, 2-oxoisocaproate, citrate and glucose, while choline and inosine were more abundant in the pHu- line. Our results highlight quantitative and qualitative differences in metabolites and nutrients potentially available to developing embryos, which could contribute to metabolic and developmental differences observed after hatching between the pHu+ and pHu- lines.

Regulation of the expression of the avian uncoupling protein 3 by isoproterenol and fatty acids in chick myoblasts: possible involvement of AMPK and PPARalpha?

The avian uncoupling protein 3 (UCP3), mainly expressed in muscle tissue, could be involved in fatty acid (FA) metabolism, limitation of reactive oxygen species production, and/or nonshivering thermogenesis. We recently demonstrated that UCP3 mRNA expression was increased by isoproterenol (Iso), a ß-agonist, in chicken Pectoralis major. This upregulation was associated with changes in FA metabolism and variations in the activation of AMP-activated protein kinase (AMPK) and in the expression of the transcription factors peroxisome proliferator-activated receptor (PPAR)α, PPARß/δ, and PPARγ coactivator-1α (PGC-1α). The aim of the present study was to elucidate the mechanisms involving AMPK and PPARα in UCP3 regulation in primary cultures of chick myoblasts. Avian UCP3 mRNA expression, associated with p38 mitogen-activated protein kinase (p38 MAPK) activation, was increased by Iso and/or FAs. The PKA pathway mediated the effects of Iso on UCP3 expression. FA stimulation also led to AMPK activation. Furthermore, the direct involvement of AMPK on UCP3 regulation was shown by using 5-aminoimidazole-4-carboxyamide ribonucleoside and Compound C. The use of the p38 MAPK inhibitor SB202190, which was associated with AMPK activation, also dramatically enhanced UCP3 mRNA expression. Finally the PPARα agonist WY-14643 strongly increased UCP3 mRNA expression. This study highlights the control of UCP3 expression by the ß-adrenergic system and FA in chick myoblasts and demonstrates that its expression is directly regulated by AMPK and by PPARα. Overexpression of avian UCP3 might modulate energy utilization or limit oxidative stress when mitochondrial metabolism of FA is triggered by catecholamines.

Early Growth and Protein-Energy Metabolism in Chicken Lines Divergently Selected on Ultimate pH.

In chickens, a divergent selection on the Pectoralis major pHu allowed the creation of the pHu+ and pHu- lines, which represent a unique model for studying the biological control of carbohydrate storage in muscle. The present study aimed to describe the early mechanisms involved in the establishment of pHu+ and pHu- phenotypes. At hatching, pHu+ chicks were slightly heavier but exhibited lower plasma glucose and triglyceride and higher uric acid. After 5 days, pHu+ chicks exhibited higher breast meat yield compared to pHu- while their body weight was no different. At both ages, in vivo muscle glycogen content was lower in pHu+ than in pHu- muscles. The lower ability of pHu+ chicks to store carbohydrate in their muscle was associated with the increased expression of SLC2A1 and SLC2A3 genes coding glucose transporters 1 and 3, and of CS and LDHα coding key enzymes of oxidative and glycolytic pathways, respectively. Reduced muscle glycogen content at hatching of the pHu+ was concomitant with higher activation by phosphorylation of S6 kinase 1/ribosomal protein S6 pathway, known to activate protein synthesis in chicken muscle. In conclusion, differences observed in muscle at slaughter age in the pHu+ and pHu- lines are already present at hatching. They are associated with several changes related to both carbohydrate and protein metabolism, which are likely to affect their ability to use eggs or exogenous nutrients for muscle growth or energy storage.

Methionine deprivation regulates the S6K1 pathway and protein synthesis in avian QM7 myoblasts without activating the GCN2/eIF2 alpha cascade.

Amino acids modulate mRNA translation through the 70 kDa ribosomal protein S6 kinase (S6K1) and the general control nondepressible 2 protein kinase (GCN2)/eukaryotic initiation factor 2 alpha eIF2 alpha pathways. The aim of the present study was therefore to explore the signaling cascades potentially modulated by methionine availability in quail muscle QM7 myoblasts using media providing all other amino acids. Methionine deprivation caused a lower S6K1 phosphorylation compared with control (Ctl) cells. Supplying the methionine-deprived media with L- and DL-methionine isomers restored S6K1 phosphorylation to the levels observed in Ctl cells. Methionine also regulated downstream S6K1 targets (i.e. ribosomal protein S6 and eukaryotic elongation factor 2), modulated translation preinitiation complex (PIC) assembly, and stimulated protein synthesis. Replacing the lacking methionine with D-methionine or its hydroxyanalog [2-hydroxy-(4-methylthio) butanoic acid] did not restore S6K1 activation or protein synthesis. Conversely, the S6K1 pathway was activated by a methionine precursor, the ketoanalog of methionine. Methionine availability regulated the GCN2/eIF2 alpha pathway. However, our results indicate that methionine deprivation led to lower protein synthesis without activating eIF2 alpha phosphorylation, a process known to limit the formation of the 43S PIC. Using the amino acid alcohol methioninol did not decrease S6K1 phosphorylation or activity and did not alter the regulation of protein synthesis by methionine. These findings suggest that methionine exerts an effect on S6K1 signaling and protein synthesis in avian QM7 myoblasts through a mechanism partly independent of the global regulation via tRNA charging.

A grape seed extract maternal dietary supplementation in reproductive hens reduces oxidative stress associated to modulation of plasma and tissue adipokines expression and improves viability of offsprings.

In reproductive hens, a feed restriction is an usual practice to improve metabolic and reproductive disorders. However, it acts a stressor on the animal. In mammals, grape seed extracts (GSE) reduces oxidative stress. However, their effect on endocrine and tissue response need to be deepened in reproductive hens. Here, we evaluated the effects of time and level of GSE dietary supplementation on growth performance, viability, oxidative stress and metabolic parameters in plasma and metabolic tissues in reproductive hens and their offsprings. We designed an in vivo trial using 4 groups of feed restricted hens: A (control), B and C (supplemented with 0.5% and 1% of the total diet composition in GSE since week 4, respectively) and D (supplemented with 1% of GSE since the hatch). In hens from hatch to week 40, GSE supplementation did not affect food intake and fattening whatever the time and dose of supplementation. Body weight was significantly reduced in D group as compared to control. In all hen groups, GSE supplementation decreased plasma oxidative stress index associated to a decrease in the mRNA expression of the NOX4 and 5 oxidant genes in liver and muscle and an increase in SOD mRNA expression. This was also associated to decreased plasma chemerin and increased plasma adiponectin and visfatin levels. Interestingly, maternal GSE supplementation increased the live body weight and viability of chicks at hatching and 10 days of age. This was associated to a decrease in plasma and liver oxidative stress parameters. Taken together, GSE maternal dietary supplementation reduces plasma and tissue oxidative stress associated to modulation of adipokines without affecting fattening in reproductive hens. A 1% GSE maternal dietary supplementation increased offspring viability and reduced oxidative stress suggesting a beneficial transgenerational effect and a potential use to improve the quality of the progeny in reproductive hens.