Metabolomics Reveals Glycerophospholipids, Peptides, and Flavonoids Contributing to Breast Meat Flavor and Benefit Properties of Beijing-You Chicken.

Unique metabolites contribute to the performance of meat flavor and potential function. In this study, UHPLC-Q Exactive HF-X-based metabolomics and multivariate analysis were applied to explore the characteristic metabolites in the breast meat of Beijing-You chicken (BYC) aged 150, 300, and 450 days (D150, D300, and D450). Based on the criteria of variable importance in the projection (VIP) > 1 and p < 0.05, a total of 154 and 97 differential metabolites (DMs) were screened out compared with D450 (D450 vs. D150, D450 vs. D300), respectively. In general, the relative content of carnosine, L-L-homoglutathione, demethyloleuropein, neohesperidin dihydrochalcone, 7-chloro-2-(3,4-dimethoxyphenyl)-3,5-dihydroxy-6,8-dimethoxy-4H-chromen-4-one, glycerophospholipids, exhibited the highest abundance at D450, while balenine, anserine, L-beta-aspartyl-L-leucine, glutathione, oxidized glutathione, stearoylcarnitine, ganoderic acid alpha, oleuroside, Lysoglycerophospholipid species (LGP) presented a downward trend with age. These 210 DMs were involved in 10 significantly enriched pathways related to the synthesis and metabolism of amino acids, peptides, and glycerophospholipid, such as glutathione metabolism, histidine metabolism, glycerophospholipid metabolism, arginine biosynthesis, tyrosine metabolism, and lysine degradation. In conclusion, this work could not only facilitate a better understanding of the differences of chicken flavor and benefit properties with age, but also provide potential valuable bioactive compounds for further research.

Dietary Crude Protein and Lysine Levels Affect Meat Quality and Myofiber Characteristic of Slow-Growing Chicken.

This study aimed to investigate the effects of dietary crude protein (CP) and lysine levels on growth performance, slaughter performance, meat quality, and myofiber characteristics of slow-growing chicken. A 3 × 3 factorial experiment was arranged, and the chickens were fed with 3 levels of dietary CP (16.0%, 17.0%, 18.0%) and 3 levels of dietary lysine (0.69%, 0.84%, 0.99%). A total of 540 8-week-old Beijing-You Chicken (BYC) female growing chickens were randomly allocated to 9 groups, 5 replicates per group, and 12 chickens per replicate. The birds were randomly allocated to one of the 9 experimental diets. Growth performance, slaughter performance, meat quality, and myofiber characteristics were determined at 16 weeks of age. The results showed that dietary CP level and the interaction of dietary CP and lysine levels affected average feed intake (AFI) (p < 0.05). The AFI in the 16.0% CP and 17.0% CP groups was higher than in the 18.0% CP group (p < 0.05). Dietary CP levels significantly affected body weight gain (BWG) (p < 0.05) at 9 to 16 weeks. The 18.0% CP group had the highest BWG (93.99 g). Dietary CP levels affected the percentage of leg muscle yield, and the percentage of leg muscle yield of the 16.0% CP group was significantly lower than that in the other two groups (p < 0.05). Dietary CP and lysine levels alone and their interactions did not affect pH24h, drip loss, and cooking loss of breast muscle (p > 0.05). The shear force of the 18.0% CP group (29.55 N) was higher than that in the other two groups (p < 0.01). Dietary CP level affected myofiber characteristic (p < 0.01), with the lowest myofiber density (846.35 p·mm-2) and the largest myofiber diameter (30.92 µm) at 18.0% CP level. Dietary lysine level affected myofiber diameter, endomysium thickness, perimysium thickness (p < 0.01), with the largest myofiber diameter (29.29 µm) obtained at 0.84% lysine level, the largest endomysium thickness (4.58 µm) at 0.69% lysine level, and the largest perimysium thickness (9.26 µm) at 0.99% lysine level. Myofiber density was negatively correlated with myofiber diameter and endomysium thickness (R = -0.883, R = -0.523, p < 0.01); perimysium thickness had a significant negative correlation with shear force (R = -0.682, p < 0.05). Therefore, reducing dietary CP level and adding appropriate lysine can reduce myofiber diameter and increase perimysium thickness, reducing shear force and improving meat tenderness. A high lysine level (0.99%) in the low-CP (16.0%) diet can improve meat tenderness by regulating the myofiber characteristic without affecting production performance.

The detection and elimination of flavin-containing monooxygenase 3 gene T329S mutation in the Beijing You chicken.

In this study, using a newly developed TaqMan-based real-time PCR method for the T329S mutation in the flavin-containing monooxygenase 3 (FMO3) gene, a marker-assisted selective breeding program against the unfavorable T allele was implemented in the Beijing You chicken breeding stock from 2010 to 2012. A total of 2,359 breeder candidate chickens were detected. After 1-generation culling in both males and females and 2-generation culling only in males, genotyping results in 2013 showed that there still remained a low unfavorable allele frequency of 0.022 in this population. The results indicated that to ensure a complete eradication of the defective tainting mutation in FMO3 out of the Beijing You population, more strict breeding and management schemes should be carried out in the future.

Proteomic Profiling of Thigh Meat at Different Ages of Chicken for Meat Quality and Development.

Chicken age contributes to the meat characteristics; however, knowledge regarding the pathways and proteins associated with meat quality and muscle development are still scarce, especially in chicken thigh meat. Hence, the objective of this study was to elucidate the intricate relationship between these traits by liquid chromatography mass spectrometry at three different ages. A total of 341 differential expressed proteins (DEPs) were screened out (fold change ≥ 1.50 or ≤0.67 and p < 0.05) among 45 thigh meat samples (15 samples per age) of Beijing-You chicken (BYC), collected at the age of 150, 300, or 450 days (D150, D300, and D450), respectively. Subsequently, based on the protein interaction network and Markov cluster algorithm (MCL) analyses, 91 DEPs were divided into 26 MCL clusters, which were associated with pathways of lipid transporter activity, nutrient reservoir activity, signaling pathways of PPAR and MAPK, focal adhesion, ECM-receptor interaction, the cell cycle, oocyte meiosis, ribosomes, taurine and hypotaurine metabolism, glutathione metabolism, muscle contraction, calcium signaling, nucleic acid binding, and spliceosomes. Overall, our data suggest that the thigh meat of BYC at D450 presents the most desirable nutritional value in the term of free amino acids (FAAs) and intramuscular fat (IMF), and a series of proteins and pathways associated with meat quality and development were identified. These findings also provide comprehensive insight regarding these traits across a wide age spectrum.

Effects of Age on Compounds, Metabolites and Meat Quality in Beijing-You Chicken Breast Meat.

The physical properties, free amino acids, and metabolites of Beijing-You chicken (BYC) breast meat aged 90, 120, and 150 days were analyzed to investigate the flavor changes with age. The shear force and intramuscular fat increased from 90 to 120 days significantly. The contents of total free amino acids and essential amino acids decreased from 90 to 120 days significantly. No significant differences were detected between 120 and 150 days. The contents of sweet amino acids, bitter amino acids, and umami amino acids showed no significant differences between different ages. In addition, GC-MS and LC-MS were integrated for metabolite detection in breast meat. A total of 128, 142, and 88 differential metabolites were identified in the comparison groups of 120 d vs. 90 d, 150 d vs. 90 d, and 150 d vs. 120 d. Amino acids and lipids were the main differential metabolites. The pathway analysis showed that arginine biosynthesis, histidine metabolism, purine metabolism, and cysteine and methionine metabolism were the main pathways involved in flavor formation during BYC development. It was also found that the metabolites associated with flavor, such as methionine, cysteine, glucose, anserine, arachidonic acid, and glycerol 1-phosphate, were significantly affected by age.

Integration of GC-MS and LC-MS for metabolite characteristics of thigh meat between fast- and slow-growing broilers at marketable age.

Thigh meat from Cobb 500 broiler (Cobb) and Beijing-You chicken (BYC) at marketable age were compared to explore the impact of growth rate on meat quality. BYC had a higher water capacity, as well as lower shear forces and IMF content than Cobb, indicating that the growth rate has a significant influence on meat quality. Additionally, 144 upregulated and 44 downregulated differential metabolites were identified using gas chromatography mass spectroscopy (GC-MS) and liquid chromatography mass spectrometer (LC-MS). Moreover, we found a significant difference between the dominant expression of metabolites in the two genotypes. The highly expressed metabolites in Cobb were amino acids and their derivatives, including l-citrulline, dimethylglycine, 1,4-butanediamine, alanine, and creatine. In contrast, BYC deposited more bioactive compounds, such as, α-linolenic acid, linoleic acid, eicosapentaenoic acid, anserine, and taurine. These results indicate that meat quality and metabolite profiles differ greatly between fast- and slow-growing chickens.

Breast Meat Fatty Acid Profiling and Proteomic Analysis of Beijing-You Chicken During the Laying Period.

The disparity in fatty acids (FA) composition exhibits a significant impact on meat quality, however, the molecular regulatory mechanisms underlying this trait in chicken are far from clear. In this study, a total of 45 female Beijing-You chicken (BYC) hens, fed on the same diet, were collected at the slaughter age of 150, 300, or 450 days (D150, D300, and D450) from sexual maturation stage to culling stage (15 birds per age). Gas chromatography-mass spectrometry (GC-MS) and tandem mass tag labeling technology based on liquid chromatography mass spectrometry (TMT-LC-MS/MS) analysis strategies were applied to profile FA compositions and to compare differential expressed proteins (DEPs) between these different slaughter ages, respectively. The FA profiling showed that increasing hen ages resulted in increased contents of both saturated and unsaturated fatty acids. Proteomic analyses showed a total of 4,935 proteins in chicken breast muscle with the false discovery rate (FDR) < 1% and 664 of them were differentially expressed (fold change > 1.50 or < 0.67 and P < 0.01). There were 410 up- and 116 down-regulated proteins in D150 vs. D300 group, 32 up- and 20 down-regulated in D150 vs. D450 group, and 72 up- and 241 down-regulated in D300 vs. D450 group. A total of 57 DEPs related to FA/lipid-related metabolisms were obtained according to the enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). These DEPs were involved in 21 significantly enriched (P < 0.05) pathways, including well-known pathways for FA synthesis (metabolism, desaturation, and elongation) and the signaling pathways for lipid metabolism (PPAR, adipocytokine, calcium, VEGF, MAPK, and Wnt). In addition, there existed several representative DEPs (FABP, FABP3, apoA-I, apoA-IV, apoC-III, apoB, VTG1, and VTG2) involved in the regulation of FA/lipid transportation. The construction of the interaction networks indicated that HADH, ACAA2, HADHA, ACSL1, CD36, CPT1A, PPP3R1, and SPHK1 were the key core nodes. Finally, eight DEPs were quantified using parallel reaction monitoring (PRM) to validate the results from TMT analysis. These results expanded our understanding of how the laying age affects the FA compositions and metabolism in hen breast meat.

UHPLC-QTOF/MS-based comparative metabolomics in pectoralis major of fast- and slow-growing chickens at market ages.

The molecular regulatory mechanism underlying meat quality between different chicken genotypes remains elusive. This study aimed to identify the differences in metabolites and pathways in pectoralis major (breast muscle) between a commercial fast-growing chicken genotype (Cobb500) and a slow-growing Chinese native chicken genotype (Beijing-You chickens, BYC) at market ages respectively based on ultra-high-performance liquid chromatography-quadrupole/time of flight mass spectrometry (UHPLC-QTOF/MS). Eighteen metabolites were identified as potential biomarkers between BYC and Cobb500 at market ages. Among them, L-cysteine exhibited a higher relative intensity in BYC compared with Cobb500 and was enriched into 10 potential flavor-associated KEGG pathways. In addition, the glycerophospholipid metabolism pathway was found to be associated with chicken meat flavor and the accumulation of sn-glycerol 3-phosphate and acetylcholine was more predominant in BYC than that in Cobb500, which were catalyzed by glycerophosphocholine phosphodiesterase (GPCPD1, EC:3.1.4.2), choline O-acetyltransferase (CHAT, EC:2.3.1.6), and acetylcholinesterase (ACHE, EC:3.1.1.7). Overall, the present study provided some metabolites and pathways for further investigating the roles of the differences in meat flavor quality in breast muscle between Cobb500 and BYC at market ages.

Comprehensive Proteomic Characterization of the Pectoralis Major at Three Chronological Ages in Beijing-You Chicken.

Chronological age is one of the important factors influencing muscle development and meat quality in chickens. To evaluate the protein expression profiles during skeletal muscle development, we performed a tandem mass tag (TMT)-based quantitative proteomic strategy in pectoralis major (breast muscle) of Beijing-You chicken (BYC) at the chronological age of 90, 120, and 150 days. Each chronological age contained 3 pooling samples or 15 birds (five birds per pooling sample). A total of 1,413 proteins were identified in chicken breast muscle with FDR < 1% and 197 of them were differentially expressed (fold change ≥1.2 or ≤0.83 and p < 0.05). There were 110 up- and 71 down-regulated proteins in 120 d vs 90 d group, 13 up- and 10 down-regulated proteins in 150 d vs 120 d group. The proteomic profiles of BYC at 120 d were very similar to those at 150 d and highly different from those at 90 d, suggesting that 120 d might be an important chronological age for BYC. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these differentially expressed proteins were mainly involved in the pathway of glycolysis/gluconeogenesis, adrenergic signaling in cardiomyocytes, focal adhesion, oocyte meiosis and phagosome. Furthermore, some DEPs were quantified using parallel reaction monitoring (PRM) to validate the results from TMT analysis. In summary, these results provided some candidate protein-coding genes for further functional validation and contribute to a comprehensive understanding of muscle development and age-dependent meat quality regulation by proteins in chickens.

Association of SNP rs80659072 in the ZRS with polydactyly in Beijing You chickens.

The Beijing You chicken is a Chinese native breed with superior meat quality and a unique appearance. The G/T mutation of SNP rs80659072 in the Shh long-range regulator of GGA2 is highly associated with the polydactyly phenotype in some chicken breeds. In the present study, this SNP was genotyped using the TaqMan detection method, and its association with the number of toes was analyzed in a flock of 158 birds of the Beijing You population maintained at the Beijing Academy of Agriculture and Forestry Sciences. Furthermore, the skeletal structure of the digits was dissected and assembled in 113 birds. The findings revealed that the toes of Beijing You chickens were rich and more complex than expected. The plausible mutation rs80659072 in the zone of polarizing activity regulatory sequence (ZRS) in chickens was an essential but not sufficient condition for polydactyly and polyphalangy in Beijing You chickens. Several individuals shared the T allele but showed normal four-digit conformations. However, breeding trials demonstrated that the T allele could serve as a strong genetic marker for five-toe selection in Beijing You chickens.

Effects of dietary L-carnitine and coenzyme Q10 at different supplemental ages on growth performance and some immune response in ascites-susceptible broilers.

Effects of dietary L-carnitine and coenzyme Q10 (CoQ10) at different supplemental ages on performance and some immune response were investigated in ascites-susceptible broilers. A 3 x 2 x 2 factorial design was used consisting of L-carnitine supplementation (0, 75, and 100 mg/kg), CoQ10 supplementation (0 and 40 mg/kg) and different supplemental ages (from day 1 on and from day 10 on). A total of 480 one-day-old Arbor Acre male broiler chicks were randomly allocated to 12 groups, every group had five replicates, each with eight birds. The birds were fed a corn-soybean based diet for six weeks. From day 10-21, all the birds were exposed to a low ambient temperature (12-15 degrees C) to increase the susceptibility to ascites. No significant effects were observed on growth performance by L-carnitine, CoQ10 supplementation, and different supplemental ages. Packed cell volume was significantly decreased by L-carnitine supplementation alone, and ascites heart index and ascites mortality were decreased by L-carnitine, CoQ10 supplementation alone, and L-carnitine + CoQ10 supplementation together (p < 0.05). Heart index of broilers was significantly improved by L-carnitine, CoQ10 supplementation alone during 0-3 week. Serum IgG content was improved by L-carnitine supplementation alone (p < 0.05), but lysozyme activity was increased by L-carnitine + CoQ10 supplementation together (p < 0.05). A significant L-carnitine by supplemental age interaction was observed in lysozyme activity. L-carnitine supplementation alone had no effects on the peripheral blood lymphocyte (PBL) proliferation in response to concanavalin A (ConA) and lipopolysaccharide, but supplemental CoQ10 alone and L-carnitine+ CoQ10 together decreased the PBL proliferation in response to ConA (p < 0.05). The present study suggested that L-carnitine + CoQ10 supplementation together had positive effects on some immune response of ascites-susceptible broilers, which might benefit for the reduction of broilers' susceptibility to ascites.

Effects of dietary L-carnitine and coenzyme Q10 supplementation on performance and ascites mortality of broilers.

The study was conducted to determine the effects of dietary L-carnitine and coenzyme Q10 (CoQ10) supplementation on growth performance and ascites mortality of broilers. A 3 x 3 factorial arrangement was employed with three levels (0, 75 and 150 mg/kg) of L-carnitine and three levels of CoQ10 (0, 20 and 40 mg/kg) supplementation during the experiment. Five hundred and forty one-day-old Arbor Acre male broiler chicks were randomly allocated into nine groups with six replicates each. All birds were fed with the basal diets from day 1 to 7 and changed to the experimental diets from day 8. During day 15 to 21 all the birds were exposed to low ambient temperature (15-18 degrees C) to induce ascites. The results showed that under this condition, growth performance of broilers were not significantly affected by CoQ10 or L-carnitine + CoQ10 supplementation during week 0-3 and 0-6, but body weight gain (BWG) of broilers was significantly reduced by 150 mg/ kg L-carnitine during week 0-6. Packed cell volume (PCV) of broilers was significantly decreased by L-carnitine and L-carnitine + CoQ10 supplementation (P < 0.05). Erythrocyte osmotic fragility (EOF), ascites heart index (AHI) and ascites mortality of broilers were significantly decreased by L-carnitine, CoQ10 and L-carnitine + CoQ10 supplementation. Though no significant changes were observed in total antioxidative capability (T-AOC), total superoxide dismutase (T-SOD) was increased by L-carnitine, CoQ10 and L-carnitine + CoQ10 supplementation (P < 0.05). Malonaldehyde (MDA) content was significantly decreased by CoQ10 and L-carnitine + CoQ10 supplementation. The results indicate that dietary L-carnitine and CoQ10 supplementation reduce ascites mortality of broilers; the reason may be partially associated with their antioxidative effects.