The Research On The Physiology Of The Death Of Myocytes In Beef During Postmortem Conditioning

After animal bleeding, all cells will be in anoxia and will receive no more nutriments. In such conditions, calcium ion will sharly elevate; ATP-related compounds will degradate; the pH value will fall down during storage.Under these unfavourable environmental modifications, muscle cells will be engaged in a cell death program, but the program is of concern in postmortem muscle and the influencere of the program on the cellular structures and meat quality main unknown. The phase in that cell death processes and the biochemical and structural changes in dying cells also main unkown. The solution for these issues is beneficial to understand the mechanisms the conversion of muscle into meat. In an effort to further, this study was conducted to explore the changes of cellular environment, verify the cell death program, quantify the level of apoptosis and understand the mechanism of the activating process of caspases-3 in three skeletal muscles of bulls during seven days postmortem storage.1. The expression of bcl-2 family proteins and the postmortem changes of energy level in myocytesThere is significant difference in fiber type constitution among LM, STN and PM muscle; with a highest slow fiber ratio found in PM muscle, followed by LM and a very low expression was displayed in STN muscles. The 35kDa GAPDH protein,26kDa bcl-2 protein,22kDa bax protein and 23kDa bad protein were detected in whole muscle lysates by SDS-PAGE and immunoblotting with specific antibodies. There was significant lower expression of bax and bcl-2 proteins in STN muscle than the ones in the PM muscle (p<0.05), while bad protein was more expressed in STN muscle than in the PM muscle; with a highest value found in STN muscle, followed by LM and a very low expression was displayed in PM muscles (p<0.05). There was a significant decrease of ATP contents from the first thirty minutes to the third hour and from the eighth to the fourteenth hour in PM and LM muscles(p<0.0001); the ATP contents in STN muscle declined from the first thirty minutes to the fifth hour and from the eighth to the fourteenth hour (p<0.0001). The difference in fiber type constitution and bad, bax and bcl-2 level among LM, STN and PM muscle could be associated with the postmortem cytochrome c release, the activation of caspases and the occurance of apoptosis. The rapid decrease in pH values in muscles could have contributed to the activation of caspases and the occurance of apoptosis; while the degradation of ATP could inhibit the activation of caspases.2. The morphological changes and the quatity of apoptosis in myocytesDuring postmortem storage, the mitochondrial morphology became to be swollen, unintact and condensated; the condensation and margination of nuclear chromatin appeared on the first day; at the fourth day postmortem, nuclear membrane displays the irregular shape characteristic of apoptosis with smaller particles packaged into similar apoptotic bodies; at the seventh day postmortem, highly electron- dense residual nuclear chromatin suggested that typical features of secondary necrosis took place. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) positive nuclei were detected at the fourth day, and increased subsequently. The count of TUNEL positive nuclei was different significantly in three muscles at the seventh day (p<0.001); with a highest TUNEL positive nuclei found in PM muscle, followed by STN and a very low expression was displayed in LM muscles. The results in HE, ACP and trypan blue staining shown that necrosis appeared in postmortem muscles of bulls; it demonstrated that it is not an unanimous program in the proteolysis of cytoskeletal and other biochemical reaction.3. The postmortem activation of caspase-3 and the pathwayWe detected 14kDa cytochrome c, active caspase-8,-9 and-3 and 36 and 24kDa cleaved PARP fragment; cytochrome c level in cytosol increased 4.5 (p<0.0001),7.9 (p<0.0001) and 7.9 (p<0.0001) times in LM, STN and PM muscles; there was higher caspase-3,-8 and -9 activities at the fourth hour than at the postmortem first thirty minutes; caspase-8 and -9 activities positively correlated to the reported caspases-3 activities across the whole storage time in muscles. The results indicated that the postmortem activation of caspase-3 appears to be mediated by both caspase-9 and caspase-8 pathways; the cleavage of PARP was the results affected by calpain and caspases-3. 4. The fiber contraction and the changes of meat qualityThe decrease of shear force was significant in PM, LM and STN muscles (p<0.01, <0.01 and <0.05, respectively) until fourth day, with a percent decline of 61.8,41.4 and 52.8% in PM, STN and LM muscles, respectively; there is a significant increase in MFI values in PM, LM and STN muscles (p<0.01,<0.05 and <0.01, respectively) until fourth and seventh day. The intermediate area increased at the first and second day in LM and PM muscles; sarcomere lengths contracted at the second day and myofibrillar ultrastructure was destroyed in muscles. Lightness (L*), redness (a*) and yellowness (b*) values increased at early phase postmortem in muscles; redness (a*) and yellowness (b*) values declined subsequently. The shear force of PM muscle was correlated with the activities of caspase-8 and -9 positively; the shear force values of muscles were correlated with the activities of caspase-3,-8 and -9 positively; wihle the apoptotic level was correlated with MFI values in PM and LM muscles positively. Meat aging, myocytes apoptosis and myofibrillar proteolysis happen in the same phase after animal bleeding. The acceleration of tenderization was associated with the activated caspases system and the occurance of apoptosis in bovine muscles during postmortem conditioning.