PGC-1α differentially regulates the mRNA expression profiles of genes related to myofiber type specificity in chicken

Previous studies on mammals showed that peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α) played a prominent role in regulating muscle fiber type transition and composition. However, the role of PGC-1α in chicken muscle has seldom been explored. To investigate the effect of PGC-1α on chicken skeletal muscles in this study, the PGC-1α gene was overexpressed or silenced in chicken primary myoblasts by using lentivirus, and then the effects of the PGC-1α gene overexpression and knockdown on the mRNA expression profile of genes related to myofiber type specificity were examined during fiber formation. The results showed that overexpression of PGC-1α from proliferation to differentiation was accompanied by the up-regulated expression of Pax7, MyoD, and CnAα, which was significantly(P0.01) increased after one day of transfection(1 I). The enhancement of MyoG, MEF2 c, and MyHC SM expression lagged, which was improved significantly(P0.01) after four days of transfection(1 I3 D). Overexpression of PGC-1α decreased(P0.01) the MyHC FWM expression after four days of transfection(1 I3 D), and it had no significant impact(P0.05) on the expression of CnB1, NFATc3, and MyHC FRM during myofiber formation. The effective silence(P0.01) of PGC-1α by lentivirus mediating short hairpin RNA(shRNA) was detected after four days of transfection(1 I3 D) in cultures, and the lack of its function in chicken primary myoblasts significantly(P0.01) down-regulated the expression of Pax7, MyoD, CnAα, MyoG, MEF2 c, and MyHC SM, significantly(P0.01) up-regulated the expression of MyHC FWM, and had no significant impact(P0.05) on the expression of CnB1, NFATc3, and MyHC FRM. These results indicated that the role of PGC-1α in regulating the fiber type specificity of chicken skeletal muscles might be similar to that in mammals, which interplayed with key genes related to myocyte differentiation and calcineurin signaling pathway.     

不同品种鸭胚胎期骨骼肌成肌细胞GHR和IGF 1 mRNA表达差异分析

为探讨生长激素受体（growth hormone receptor,GHR）和胰岛素样生长因子 1（Insulin like growth factor 1,IGF 1）在不同品种鸭胚胎期骨骼肌成肌细胞中的表达差异。选择生长速度不同的金定鸭和高邮鸭为试验动物,采用实时荧光定量PCR方法检测鸭13,15,17,19,21和23胚龄时胸肌和腿肌成肌细胞GHR,IGF 1 mRNA的表达情况,并进行了品种间比较。结果发现：13胚龄时,高邮鸭胸肌和腿肌成肌细胞IGF 1的表达均显著高于金定鸭（P<0.05）;17胚龄是两个品种鸭GHR和IGF 1共同的表达高峰期;17胚龄之后,鸭胸肌和腿肌成肌细胞GHR和IGF 1表达均显著降低。在19,21胚龄时,高邮鸭胸肌成肌细胞2个基因的表达均显著高于金定鸭（P<0.05）,而腿肌成肌细胞GHR和IGF 1表达在品种间无显著差异（P>0.05）。鸭胚胸肌和腿肌成肌细胞GHR和IGF 1表达呈现极显著的正线性相关（P<0.01）。提示：GHR和IGF 1的表达谱及各自在品种间的变化规律基本一致;鸭胚骨骼肌成肌细胞GHR和IGF 1 mRNA表达有组织特异性,二者之间可能存在正调控的作用机制。     

Effect of MSTN Propeptide and shRNA Co-expression Vector on Proliferation of Skeletal Muscle Satellite Cells

Myostatin(MSTN)is a negative regulator of skeletal muscle growth,in order to study the effect of inhibition MSTN expression on the proliferation of bovine skeletal muscle satellite cells,we constructed co-expression vector pcDNA3.1-ProMSTNshRNA,transfected it into muscle satellite cells by Liposome 2000,and detected cell proliferation changes by CCK-8 method and flow cytometry after 48 h.The expressions of P21 and CDK2 were detected by Western blot and real-time PCR.The results showed that the cell vitality of experimental groups significantly increased than that of the negative control,and cells in S phase also increased significantly(P<0.05).After knocked down MSTN gene,P21 expression decreased(P<0.05),but CDK2 gene expression increased(P<0.05).These results indicated that MSTN gene expression was associated with P21 and CDK2,the proliferation of skeletal muscle satellite cells could be promoted while MSTN was inhibited,which provided a theoretical basis for the study on transgenic cattle.     

Effects of dietary forage to concentrate ratio and wildrye length on nutrient intake,digestibility, plasma metabolites,ruminal fermentation and fecal microflora of male Chinese Holstein calves

Twenty-eight male, weaned Chinese Holstein calves((156.8±33.4) kg) were used to investigate the effects of dietary forage to concentrate ratio(F:C) and forage length on nutrient digestibility, plasma metabolites, ruminal fermentation, and fecal microflora. Animals were randomly allocated to four treatments in a 2×2 factorial arrangement: whole-length forage(WL) with low F:C(50:50); WL with high F:C(65:35); short-length forage(SL) with high F:C(65:35); and SL with low F:C(50:50). Chinese wildrye was used as the only forage source in this trial. The grass in the SL treatments was chopped using a chaff cutter to achieve small particle size(~50% particles 19 mm). Dry matter intake(DMI) and organic matter(OM) intake was increased by increasing both F:C(P0.01) and forage length(FL)(P0.05), while acid detergent fiber(ADF) and neutral detergent fiber(NDF) intakes were only increased by increasing the F:C(P0.01). The digestibility of NDF was increased as the FL increased(P0.01), and it was also affected by interaction between F:C and FL(P0.05). Cholesterol(CHO)(P0.01), leptin(LP)(P0.05), and growth hormone(GH)(P0.01) concentrations in plasma were increased as dietary F:C increased. A significant increase in plasma triglyceride(TG)(P0.01), insulin(INS)(P0.05), and GH(P0.01) levels was observed with decreasing dietary FL. Ruminal p H values of calves fed with low F:C diets were significantly lower than those in high F:C treatment(P0.05). Increasing the F:C enhanced ruminal acetic acid(P0.05) and acetic acid/propionic acid(P0.01). Fecal Lactobacillus content was significantly higher, while Escherichia coli and Salmonella contents were significantly lower in WL and high F:C groups(P0.05). Lower fecal scores(higher diarrhea rate) were observed in calves fed with SL hay compared to WL hay(P0.05). Denatured gradient gel electrophoresis(DGGE) bands and richness index(S) were significantly affected by the interaction between F:C and FL(P0.05), under high F:C, band numbers and richness index from WL group were higher than that from SL group(P0.05), whereas there were no differences between WL andSL groups under low F:C(P0.05). Microflora similarity was 50–73% among the different treatments. It is concluded that the WL with high F:C(65:35) diet is suitable for weaned calves.     

Effect of relative humidity at either acute or chronic moderate temperature on growth performance and droppings' corticosterone metabolites of broilers

The present study aimed to investigate the effect of relative humidity(RH) at either acute or chronic moderate ambient temperature(Ta) on growth performance and droppings' corticosterone metabolites of broilers.Two experiments were conducted: effect of RH(35,60 or 85%) on average daily feed intake(ADFI) and droppings' corticosterone metabolites at acute(1 d: 20–26 or 31–20°C,26 or 31°C for 6 h d–1 at 10:00–16:00) moderate Ta(experiment 1) and effect of RH(35,60 or 85%) on growth performance and droppings' corticosterone metabolites at chronic(step-wisely increasing temperature by 3°C every 3 d from 20 to 32°C within 15 d: 20–23–26–29–32°C) moderate Ta(experiment 2).Droppings were collected at the 2,4,6,8,and 22 h after Ta-RH controlled in experiment 1 and at the 2,4,6,and 22 h after Ta controlled to 32°C in experiment 2.The results showed that: 1) In experiment 1,85% RH increased(P0.05) the droppings' corticosterone metabolites at the 2,6,8,and 22 h and 35% RH increased(P0.05) it at the 2 and 22 h compared to the 60% RH.Moreover,85% RH further increased(P0.05) it compared to the 35% RH,however,no difference(P0.05) was found in ADFI among the three RH groups at acute moderate 26°C; 35 and 85% RH increased(P0.05) droppings' corticosterone metabolites at the 2,6,8 and 22 h and decreased(P0.05) ADFI compared to the 60% RH,moreover,85% RH further increased(P0.05) droppings' corticosterone metabolites and further decreased(P0.05) ADFI compared to the 35% RH at acute moderate 31°C; and the average of droppings' corticosterone metabolites in the whole period had a negative correlation(P0.02) with the ADFI.2) In experiment 2,85% RH increased(P0.01) droppings' corticosterone metabolites only at the 2 h and decreased(P0.02) ADFI and average daily gain(ADG) compared to the 60% RH,no difference(P0.05) in droppings' corticosterone metabolites was found between the 35 and 60% RH,however,35% RH decreased(P0.01) ADG compared to the 60% RH,and the average of droppings' corticosterone metabolites in the whole period also had a negative correlation(P0.02) with ADFI and ADG.In conclusion,droppings' corticosterone metabolites could be used as a RH stress index and low and high RH,especially high RH,reduced growth performance possibly through inducing RH stress at moderate temperature.     

Analysis of DNA methylation of CD79B in MDV-infected chicken spleen

Marek's disease (MD), an immunosuppressive disease induced by Marek's disease virus (MDV), provides an ideal model for studying diseases caused by a carcinogenic virus. CD79B is a B-cell antigen receptor complex-associated protein β-chain precursor which is involved in the activation, proliferation, differentiation of B-cell and the transmission of downstream signals. This study analyzed CD79B gene mRNA expression and methylation by two schemes #20 (5´ flanking to intron 1) and #27 (intron 2 to intron 3), between MDV-infected tumorous spleens (TS) and non-infected spleens (NS). Results showed that average methylation levels of CpGs in #20 and #27 were higher in TS than in NS (P<0.05), while, CD79B mRNA expression was lower in TS than in NS (P<0.01). Six of 40 CpG sites showed significantly (P<0.05) different methylation levels between TS and NS. Correlation analysis showed that the average methylation level rather than a single site methylation level in #20 affected (P<0.05) mRNA expression. Collectively, it was found that the change of CD79B gene expression after MDV infection might be partly explained by modification of DNA methylation.     

Correlation analysis of relationships between polymorphisms of high quality chicken myogenin gene and slaughter and meat quality traits

In this study, PCR-SSCP technique was designed to investigate the effect of the myogenin (MyoG) gene on quality of chicken meat (developed by Sichuan Dahen Poultry Breeding Company using local breeds). Four mutations at base position in the promoter region were detected among individuals in each line, i.e. T/C in locus A, and T/A, T/C and A/G in locus B. Least squares analysis showed that there was a significant difference between genotype and breast muscle percentage and some carcass traits (P < 0.05) for locus A. There were significant differences (P < 0.05) in breast muscle weight between AC, AA and AB genotypes; a significant difference (P < 0.05) in leg muscle percentage between CC and AC for locus B, and an extremely significant difference (P < 0.01) in the frequency of genotype muscle fiber density for both locus A and locus B. Nonsignificant difference (P < 0.05) was detected in the other traits. It was concluded that the MyoG gene is the major gene affecting the muscle fiber traits of chicken or it links with the candidate gene, and the mutation can be used as the molecular genetic marker to select the chickens for meat quality traits. __________ Translated from Hereditas (Beijing), 2007, 29(9): 1089–1096 [译自: 遗传(北京)] These authors contributed equally to this work.     

Two new IncRNAs regulate the key immune factor NOD1 and TRAF5 in chicken lymphocyte

Reticuloendotheliosis virus (REV) causes the atrophy of immune organs and immuno-suppression in chickens, but the underlying molecular mechanism of the immune response after infection by REV is not well understood. Presently, the RNA-seq was used to analyze the regulation of immune response to REV in chicken lymphocytes from peripheral blood. Overall, 134 differentially expressed long non-coding RNAs (lncRNAs) between cells with REV infection or without in vitro were screened. Based on the differentially expressed protein-coding genes, the nucleotide-binding oligomerization domain (NOD)-like receptor pathway related to immune regulation was enriched. Two lncRNAs (L11530 and L09863) were predicted to target the NOD1 and tumor necrosis factor receptor-associated factor 5 (TRAF5) gene, respectively, which are involved in the NOD-like receptor pathway with cis-regulation way. The in vitro results revealed the significantly up-regulated (P<0.01) levels of lncRNA-L11530 and its target gene, NOD1, and the significantly down-regulated (P<0.05) levels of lncRNA-L09863 and its target gene, TRAF5, in lymphocytes after REV infection. These changes also occurred in vivo in blood lymphocytes of chickens infected with REV. Further, L09863 and L11530 were respectively interfered, the expression levels of their target genes NOD1 or TRAF5 were significantly down-regulated, accompanied by the change of IL-8 and IL-18 secretions in lymphocytes. The NOD-like receptor pathway appears to be important in the immune response to REV, LncRNA-11530 and IncRNA-09863 might involve in the immune regulation on REV infection by targeting NOD1 or TRAF5 in blood lymphocytes of chickens. Our findings reveal a new regulation of lncRNAs (L11530 and L09863) on immunity in chicken peripheral blood lymphocytes for REV infection by changing the expression of the target genes via the NOD-like receptor pathway.     

Knockdown of the Meq gene in Marek's disease tumor cell line MSB1 might induce cell apoptosis and inhibit cell proliferation and invasion

Marek's disease (MD), a highly cell-associated and contagious disease of chickens caused by Marek's disease virus (MDV) can result in neural lesions, immunosuppression and neoplasia in chicken. The Meq gene is an important oncogene in the MDV genome, and it is expressed highly in MD tumor tissues and MD T-lymphoblastoid cell lines. An experiment was conducted to elucidate the role of Meq in MD tumor transformation. RNA interference technology was used to block its expression, and then analyzed the biological effects of Meq knockdown on the MD tumor cell line MSB1. A small interfering RNA with an interference efficiency of 70% (P<0.01) was transfected into MSB1 cells to knock down the expression of Meq gene. The cell proliferation, cycle and apoptosis were detected post-Meq knockdown. The results showed that MSB1 cell proliferation was downregulated remarkably at 48 h (P<0.01), 60 h (P<0.05) and 72 h (P<0.01) post-Meq knockdown. The cell cycle was unaffected (P>0.05). B-cell lymphoma 2 gene (BCL2) was anti-apoptotic and caspase-6 was the effector in the apoptosis pathway. The activity of caspase-6 was upregulated (P<0.05) significantly and BCL2 gene expression was downregulated (P<0.05) significantly post-Meq knockdown, suggesting cell apoptosis might be induced. MSB1 cell migration did not exhibit any obvious change (P>0.05) post-Meq knockdown, but the expression of two genes (matrix metalloproteinase 2 (MMP2) and MMP9) that are correlated closely to cell invasion was downregulated (P<0.05) remarkably post-Meq knockdown. The Meq knockdown might affect the main features of tumorous cells, including proliferation, apoptosis, and invasion, suggesting that the Meq gene might play a crucial role in interfering with lymphomatous cell transformation.     

Phosphorylation of sarcoplasmic and myofibrillar proteins in three ovine muscles during postmortem ageing

This study aimed to examine changes in phosphorylation of sarcoplasmic and myofibrillar proteins from longissimus lumborum, semitendinosus, and psoas major muscles during postmortem ageing for 5 d. These sarcoplasmic and myofibrillar proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with phosphorous and protein specific stains. Myofibril fragmentation index, pH, the content of lactic acid and the relative activity of μ-calpain in three ovine muscles were measured. These results showed that the relative phosphorylation level of sarcoplasmic and myofibrillar proteins of psoas major muscle were lower compared with longissimus lumborum and semitendinosus muscles (P<0.05). The pH of psoas major muscle was the lowest at 0.5 h postmortem, and the highest after 12 h postmortem (P<0.05). In addition, the relative activity of μ-calpain was higher within 5 d postmortem and myofibril fragmentation index was higher after 1 d postmortem in psoas major muscle than those of longissimus lumborum and semitendinosus muscles (P<0.05). The sarcoplasmic protein phosphorylation may regulate the rate of pH decline to influence the μ-calpain activity and then proteolysis of proteins consequently. This study gives a new perspective of the mechanism of postmortem meat tenderization.     

Effect of dioscorea opposite waste on growth performance,blood parameters,rumen fermentation and rumen bacterial community in weaned lambs

This study investigated the effects of dioscorea opposite waste(DOW) on the growth performance, blood parameters, rumen fermentation and rumen microbiota of weaned lambs. Sixty healthy weaned Small-Tailed Han lambs(male,(22.68±2.56) kg initially) were used as the experimental animals. Four levels of concentrate: 0(control, CON), 10%(DOW1), 15%(DOW2) and 20%(DOW3), were replaced with DOW in the basal diet as experimental treatments. The results showed that lambs fed the DOW2 diet had a higher(P&l...     

Effect of relative humidity at chronic temperature on growth performance,glucose consumption,and mitochondrial ATP production of broilers

An experiment was conducted to investigate the effect of relative humidity(RH) at chronic temperature on growth performance, glucose consumption, and mitochondrial adenosine triphosphate(ATP) production of broilers. A total of 180 28-day-old Arbor Acres broilers(half males and half females) were randomly allocated to three treatments, each containing six replicates of 10 birds per treatment, using a completely randomized design. Birds were reared at 35, 60 or 85% RH at 32°C for 15 days(temperature increased by 3°C every 3 days from 20 to 32°C within 15 days: 20–23–26–29–32°C). RH affected(P0.05) average daily feed intake(ADFI), average daily gain(ADG), average daily water consumption(ADWC), blood glucose concentrations, muscle glycogen levels, avian uncoupling protein(av UCP) mRNA expression, and cytochrome c oxidase(CCO) activity in liver of broilers at 42 days of age. The 85% RH decreased(P0.05) ADFI, ADG and ADWC; 35% RH decreased(P0.02) ADG. Both 85 and 35% RH increased(P0.01) blood glucose and decreased(P0.05) muscle glycogen. Both 85 and 35% RH increased(P0.05) av UCP mRNA expression. 35% RH decreased(P0.05) CCO activity. In conclusion, both high and low RH inceased glucose consumption and reduced mitochondrial ATP poduction, leading to a decline in growth rate.