Dynamic changes of blood glucose, serum biochemical parameters and gene expression in response to exogenous insulin in Arbor Acres broilers and Silky fowls.

Silky chicken is a breed of chickens with black skin and slow growth rate used in Chinese traditional medicine, whereas Arbor Acres broiler is a well-known commercial breed in the poultry industry, it is featured by a large size, rapid-growth rate, high feed-conversion rate and strong adaptability. The difference in their rate of growth may be primarily related to different mechanism for glucose metabolism. Here we compared the insulin sensitivity of the two breeds; we investigated the temporal changes (at 0 min, 120 min and 240 min) of serum insulin and other biochemical parameters and determined the spatio-temporal changes of gene mRNA abundance in response to exogenous insulin (80 µg/kg body weight). The results indicated that: (1) Silky chickens showed stronger blood glucose recovery than broilers in the insulin resistance test. (2) The serum urea level in Silky chickens was twice of broilers; exogenous insulin significantly up-regulated serum uric acid level in Silky fowls in a time-dependent manner and increased serum cholesterol content at 120 min. (3) Two breeds showed distinctly different temporal changed in serum insulin in response to exogenous insulin stimulation. The fasting serum insulin concentration of broilers was three-fold of Silky chickens at the basal state; it decreased significantly after insulin injection and the levels at 120 min and 240 min of broilers were only 23% (P < 0.01) and 14% (P < 0.01) of the basal state, respectively. Whereas the serum insulin content in Silky chickens showed stronger recovery, and the 240 min level was close to the 0 min level. (4) GLUT2, GLUT12, neuropeptide Y and insulin receptor (IR) were predominantly expressed in the liver, pectoralis major, olfactory bulb and pancreas, respectively, where these genes presented stronger insulin sensitivity. In addition, the IR mRNA level was strongly positively with the GLUT12 level. In conclusion, our findings suggested that Silky chickens have a stronger ability to regulate glucose homeostasis than broilers, owing to their higher IR levels in the basal state, stronger serum insulin homeostasis and candidate genes functioning primarily in their predominantly expressed tissue in response to exogenous insulin.

The distinct spatiotemporal distribution and effect of feed restriction on mtDNA copy number in broilers.

Mitochondrial DNA (mtDNA) copy number reflects the abundance of mitochondria in cells and is dependent on the energy requirements of tissues. We hypothesized that the mtDNA copy number in poultry may change with age and tissue, and feed restriction may affect the growth and health of poultry by changing mtDNA content in a tissue-specific pattern. TaqMan real-time PCR was used to quantify mtDNA copy number using three different segments of the mitochondrial genome (D-loop, ATP6, and ND6) relative to the nuclear single-copy preproglucagon gene (GCG). The effect of sex, age, and dietary restriction (quantitative, energy, and protein restriction) on mtDNA copy number variation in the tissues of broilers was investigated. We found that mtDNA copy number varied among tissues (P < 0.01) and presented a distinct change in spatiotemporal pattern. After hatching, the number of mtDNA copies significantly decreased with age in the liver and increased in muscle tissues, including heart, pectoralis, and leg muscles. Newborn broilers (unfed) and embryos (E 11 and E 17) had similar mtDNA contents in muscle tissues. Among 42 d broilers, females had a higher mtDNA copy number than males in the tissues examined. Feed restriction (8-21 d) significantly reduced the body weight but did not significantly change the mtDNA copy number of 21 d broilers. After three weeks of compensatory growth (22-42 d), only the body weight of broilers with a quantitatively restricted diet remained significantly lower than that of broilers in the control group (P < 0.05), while any type of early feed restriction significantly reduced the mtDNA copy number in muscle tissues of 42 d broilers. In summary, the mtDNA copy number of broilers was regulated in a tissue- and age-specific manner. A similar pattern of spatiotemporal change in response to early feed restriction was found in the mtDNA content of muscle tissues, including cardiac and skeletal muscle, whereas liver mtDNA content changed differently with age and dietary restriction. It seems that early restrictions in feed could effectively lower the mtDNA content in muscle cells to reduce the tissue overload in broilers at 42 d to some degree.

The spatio-temporal features of chicken mitochondrial ND2 gene heteroplasmy and the effects of nutrition factors on this gene.

Mitochondrial heterogeneity is the presence of two or more types of mitochondrial (mt)DNA in the same individual/tissue/cell. It is closely related to animal health and disease. ND2 is a protein-coding gene in mtDNA, which participates in mitochondrial respiratory chain and oxidative phosphorylation. In previous studies, we observed that the mt.A5703T and mt.T5727G sites in the ND2 gene were the heteroplasmic variation sites. We used pyrophosphate sequencing technology to examine chicken mt.A5703T and mt.T5727G heteroplasmic sites in the ND2 gene, in different tissues and at different development stages in chickens. We also investigated whether nutritional factors could affect the mt.A5703T and mt.T5727G heteroplasmy. Our results showed that chicken mt.A5703T and mt.T5727G heteroplasmy had clear spatio-temporal specificities, which varied between tissues/development stages. The mtDNA heterogeneity was relatively stable upon nutrition intervention, 30% dietary energy restriction (from 18 to 48 days old) and different types of dietary fats (at 5% concentration, from 1 to 42 days old) did not change the breast muscle heteroplasmy of broilers at the mt.A5703T and mt.T5727G sites. In addition, multiple potential heteroplasmic sites were detected by clone sequencing in the ND2 region, which potentially reflected abundant heteroplasmy in the chicken mitochondrial genome. These results provide an important reference for further research on heteroplasmy in chicken mitochondria.

Heteroplasmy in the complete chicken mitochondrial genome.

Chicken mitochondrial DNA is a circular molecule comprising ~16.8 kb. In this study, we used next-generation sequencing to investigate mitochondrial heteroplasmy in the whole chicken mitochondrial genome. Based on heteroplasmic detection thresholds at the 0.5% level, 178 cases of heteroplasmy were identified in the chicken mitochondrial genome, where 83% were due to nucleotide transitions. D-loop regionwas hot spot region for mtDNA heteroplasmy in the chicken since 130 cases of heteroplasmy were located in these regions. Heteroplasmy varied among intraindividual tissues with allele-specific, position-specific, and tissue-specific features. Skeletal muscle had the highest abundance of heteroplasmy. Cases of heteroplasmy at mt.G8682A and mt.G16121A were validated by PCR-restriction fragment length polymorphism analysis, which showed that both had low ratios of heteroplasmy occurrence in five natural breeds. Polymorphic sites were easy to distinguish. Based on NGS data for crureus tissues, mitochondrial mutation/heteroplasmy exhibited clear maternal inheritance features at the whole mitochondrial genomic level. Further investigations of the heterogeneity of the mt.A5694T and mt.T5718G transitions between generations using pyrosequencing based on pedigree information indicated that the degree of heteroplasmy and the occurrence ratio of heteroplasmy decreased greatly from the F0 to F1 generations in the mt.A5694T and mt.T5718G site. Thus, the intergenerational transmission of heteroplasmy in chicken mtDNA exhibited a rapid shift toward homoplasmy within a single generation. Our findings indicate that heteroplasmy is a widespread phenomenon in chicken mitochondrial genome, in which most sites exhibit low heteroplasmy and the allele frequency at heteroplasmic sites changes significantly during transmission events. It suggests that heteroplasmy may be under negative selection to some degree in the chicken.