| Meat quality includes meat color, juiciness, tenderness, flavor, of which, tenderness is an important characteristics that consumers always pay special attention to. After slaughter, oxygen supply and blood circulation is terminated, a series of changes will happen within animal body, for example, energy metabolism changes from aerobic to anaerobic ways and rigor mortis happens. Biochemical changes at early postmortem time will affect meat color, water holding capacity and tenderness, and then affect he formation of meat quality. Breakdown of myofibrillar protein dependent on the activities of proteolytic enzymes will eventually affect meat tenderness. As a part of management, pre-slaughter fasting will result in stress, and then affect energy metabolism and related biochemical changes. However, there are few data available on this field. In the present study, we investigate effects of preslaughter fasting on proteolytic enzymes, and gene expression in chicken breast muscle.1. Effect of fasting on energy metabolism and tenderizing enzymesChickens were deprived of feed for0h,12h and24h before slaughter. Blood samples were centrifuged and used for the determination of plasma corticosterone. Breast muscles were removed and cut into3parts and stored at4℃for0h,3h and10h. Samples were used for analyses of enzymes and energy metabolism. The results indicated that fasting for24h resulted in greater ultrastructural changes and plasma corticosterone levels than fasting for12h and control groups. Fasting caused the accelerated depletion of glycogen, ATP and ADP before or immediately after slaughter, but no difference existed in ATP at3and10h. Fasting resulted in greater ultimate pH value. Zymography indicated that fasting delayed the activation of μ/m-calpain, however, it accelerated the release of lysosomal enzymes. Therefore, in terms of animal welfare and meat quality, fasting for no more than12h is acceptable in practice. 2. Effect of fasting on protein expression and phosphorylation levelSarcoplasmic and myofibrillar proteins were separated on SDS-PAGE gels, and then stained by Pro-Q Diamond and Sypro Ruby stains. Fasting significantly affected phosphorylation level of sarcoplasmic proteins. The2-D electrophoresis of sarcoplasmic proteins indicated that fasting resulted in an increase in the expression and phosphorylation of heat shock protein71.Phosphorylation of PK and ENOB varied with different molecular weights and isoelectric points. Most differentially expressed proteins in sarcoplasm are glycolytic enzymes, and fasting improved their phosphorylation levels. This indicated that fasting affects muscle glycolytic processes mainly by protein phosphorylation modification. Phosphorylation level of few proteins were low after fasting24h, and long-term fasting delayed activation of some enzymes maybe the reason. Above all, fasting12h is preferred.3. Effect of fasting on gene expressionGenes of non-fasting and fasting groups immediately after slaughter were sequenced by RNA-seq. Sequencing saturation analysis, randomness assessment and the proportion of cleans reads indicated that this sequencing is reliable, and provides accurate results. Contrast between fasting groups and non-fasting group indicated that differently expressed genes mainly relate to cell components and biological processes in GO database. This means that fasting mainly affect gene expression in this two groups, and the effect on genes related to molecular function is relatively small. There are few differences between two fasting groups. After fasting, few genes related to energy metabolism were differently expressed, but energy metabolism enzymes were affected significantly. This may be due to the phosphorylation modification of energy metabolism enzymes after translation. |
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