The Mechanism Of Skeletal Muscle Protein Degradtion And Effects Of Disaccharides Administration Of Late-Term Duck Embryos

During the end of incubation, the energy reserve for growth and development is quickly depleted and the embryo may survive by "self-degradation". The present study first cloned the key genes in Autophagy and Ubiquitin proteasome pathway and analyzed the tissue expression profile; furthermore, provide exogenous disccharides to late-term duck embryos through in ovo feeding technology to examine the effect of IOF carbohydrates on energy status and skeletal muscle protein degradation; finally elucidate the underlying molecular mechanism of muscle protein degradation by in vitro experiments.In experiment 1, three healthy 25d of age ducks were killed and pectoral muscle were collected. Duck muscle cDNA were used to clone the key genes of UPP and Autophagy system. The present study successfully cloned 1068bp Atrogin-1 and 628bp MuRFl in UPP system; 299bp LC3,553bp Beclinl,753bp Atg5 in Autophagy system.Small intestine, heart, liver, pectoral muscle, gizzard, brain were isolated from the 25E and Od ducks for the study of Atrogin-1 and Atg5 tissue expression profile. Atrogin-1 was expressed highest in pectoral muscle (P<0.05), and it increased from 25E to Od in all the tissue (P<0.05). Atg5 was highly expressed in small intestine and brain. Atg5 also and it increased from 25E to Od in all the tissue (P<0.05). These results showed Atrogin-1 mediated UPP mainly happens on skeletal muscle, and autophagy is universal exist. These results also suggested protein degradation was enhanced during the end of incubation.In experiment 2, one hundred and eighty duck eggs were randomly divided into two groups, control group and IOF group. In ovo injection (2% maltose,2% sucrose and 0.35% sodium chloride) were conducted at 23E, duckling were provided water ad libitum and fed commercial duck diet at 2d of age. Ten samples from each group were collected at 22E,25E, 0d,2d,4d, and dextral pectoral muscle were weighted. The results were as forrowing:1. Muscle weight was significantly decreased at Od as compared with 22E and 25E (P<0.05), muscle protein content at Od was much lower than 22E in control group. IOF carbohydrates had no effects on muscle weights (P>0.05); but significantly improved muscle protein content at 25E (P<0.05). 2. The expression of Atrogin-1 and MuRF1 were increased as the progress of hatching and reached maximal at 2d. From 22E to 2d of age, the mRNA levels of Atrogin-1 and MuRF1 increased 126 fold and 87 fold, respectively (P<0.05). IOF significantly inhibited the expression of Atrogin-1 and MuRF1 at 2d (P<0.05). The expression of LC3 and Atg5 reached maximal at Od and 2d of age, respectively. Similarly, IOF suppressed the expression of LC3 and Atg5 at 2d of age. The relative expression level of LC3 and Atg5 were lower than Atrogin-1 and MuRF1.3. In control group, activation of AMPK were observed at 22E and 25E in muscle and liver, respectively (P<0.05). No significant effects of IOF were observed on activation of AMPK in brain and muscle, but IOF suppressed activation of AMPK in liver at 2d of age(P=0.07).These results indicated during the end of incubation energy were limited which lead to the degradation of muscle protein. UPP and AP were both involved in the muscle protein degradation, and UPP may play a dominant role in this specific situation. IOF could allievate muscle protein degradation by suppression the expression of key genes in UPP and AP system.In experiment 3, primary duck muscle cells were devided into two groups, non-starvation group and starvation group. RNAi were used to down-regulate Atrogin-1 in primary duck muscle cells. Three siRNA were designed, shRNA1, shRNA2, shRNA3. Two pairs of vectors as negative control and positive control.24h after RNAi, the culture medium of starvation group were changed to PBS. Cells were collected 30h after RNAi. The expression of Atrogin-1, MuRF1, LC3 and Atg5 were significantly increased in starvation group as compared with non-starvation group (P<0.05). In positive and negative controls, there were no significant difference of AMPK activation between starvation and normal state. However, in shRNA3 treatment, AMPK were highly activated when Atrogin-1 down-regulated (P<0.05).The present study observed muscle protein degradation in late-term duck embryo, and demonstrated it was mainly mediated by UPP. The role of muscle protein degradation by UPP was to provide energy for growth and development. IOF could partially alleviate muscle protein degradation.