The Effect Of Pre-Slaughter Stress On Meat Quality And Its Machanisms In Pigs

This paper aimed to study different pre-slaughter stress on meat quality, animal welfare as well as biochemical indicator, and its molecular mechanism at the cellular level. Firstly,120 HalNN Large White×Landrace finishing pigs were chose to study different feed restriction time (fasting 8 h and 24 h), transport plant (transport time, Short:45 min vs. Medium:3 h vs. Long:5 h; stocking densities, Low:5 pigs per pen or 230 kg/m2, Medium:6 pigs per pen or 275 kg/m2 and High:7 pigs per pen or 325 kg/m2), lairage conditions (3 h lairage group with or withou toys,0 h lairage group) and slaughter measures (directly killing group and electrical stun-kill) on pre-slaughter behavior, meat quality, blood physiological and biochemical indicators after slaughter. On the basis of animal experiments, a mechanism that the increase of cortisol and ACTH after pre-slaughter stress in the blood is a major factor to decline post-mortem meat quality is proposed. To test this hypothesis, the stress group was made up of 72 pigs from transport plan,8 pig with no lairage time and 16 pig from different slaughter measures group, and 16 pigs with or without toys under 3 h lairage were merged into control group. Analysis showed poor meat quality usually accompanies with an exaggerated release of cortisol. And then a muscle cell stress model was established by the use of mice muscle cells and primary porcine muscle cells to reveal that dexamethasone (a synthetic glucocorticoid hormone, cortisol is one of glucocorticoids) via affecting the calcium channel to determine whether the muscle cell toward survival or apoptosis, and eventually affecting the post-mortem of meat quality. The main results as follows:The results showed:①8 h feed restriction pigs exhibited no differences on cortisol, ACTH, lactate, LDH, CK and glucose when compared to 24 h group. Whereas,8 h feed restriction pigs displayed lower average values on cortisol and ACTH.②The values of EC45min and pH24 in longissimus muscle was higher after 8 h feed restriction than those feed restricted for longer time (24 h; P<0.05), while other meat quality were not affected by any treatment (P>0.05).③The pigs from the 24 h feed restriction group had significantly higher SCC, RDW-SD (P<0.05) compared with the shorter time groups, while other blood constituents were not significantly influenced by feed restriction (P> 0.05). There are still some influences on the extent of stress when pig under different fasting time, and also have significant influence on the part of meat quality and immune indicators. After considering pig welfare, meat quality, immunization indexes, economic, and the effect of intestinal contents on carcass after pigs slaughtered, we must give priority to fast for 24 h.A 3×3 factorial experiment has been conducted and combining three transport time and three stocking densities to form 9 transport pressure groups. On arrival at the slaughterhouse, the pigs were slaughtered immediately. A total of seventy two pigs have been recorded. The results showed:①LDH enzymatic activity increased as transport time increased, but it reached significance only for the high density group (P<0.05). Lactate concentration was significantly lower in the group subjected to 3 h transport and low stocking density (P<0.05). The concentration of cortisol was higher after 5 h transport than those transported for shorter time (45 min or 3 h; P<0.05) and also significantly increased (P<0.05) in the highest stocking density. The stocking density treatment had a difference tendency that ACTH concentration gradually increased as density enhanced.②Drip loss, pH24 and MC24 were significantly affected by transport time in the longissimus muscle. MC24 was the only meat quality that was affected by transport time in the semimembranosus muscle, while the values of pH45min, pH24, MC45min and MC24 were lower (P<0.05) in the low density group than medium or high density group.③The pigs from the 3 h transport group had significantly lower RBC, HGB and HCT (P<0.05) compared with the short or long time transport groups. At the same time, pigs under the long time transport showed lower count of lymphocyte than pig under 1 and 3 h transport conditions. Hence, from these data we conclude that the most adequate pre-slaughter transport time was three hours for medium stocking density (less than 275 kg/m2) and at a warm environmental temperature in the Chinese commercial transport conditions. 40 pigs were transported in advance 3h and randomly allocated into two groups to experience two lairage conditions that 20 pigs per group experienced lairage 3 h treatment with or without toys respectively, and were slaughtered together with other 20 on arrival at slaughterhouse immediately. Finally,8 pigs from every group and totally 24 were recorded in this study. The results showed:①3 h lairage group with toys demonstrated significantly higher mental performance than group without toys at 3 sampling times. All the pigs showed increasedly calm with the lairage time running off.②0 h lairage increased plasma cortisol, ACTH and lactate (P<0.05), while decreased plasma lactate dehydrogenase (LDH), creatine kinase (CK) (P<0.05). All biochemical indexes were not influenced by toys during lairage (P<0.05).③Muscle colour value, electrical conductivity, pH at 45 min and 24 h from LM and SM and drip loss were not affected by any treatment (P>0.05).④3 h lairage with toys and 3h lairage pigs exhibited high red blood cell (RBC), hemoglobin (HGB), haematocrit (HCT) when compared to 0 h lairage. Whereas,3 h lairage with toys displayed higher white blood cell (WBC) levels than 0 h lairage. In addition, Lymphocyte (W-SCC) was significantly higher in 3 h lairage compared with 0 h lairage. It didn't affect any of the hemocyte indexes between 3 h lairage with and without joys. We could conclude on the basis of the results obtained from this pilot study that in local commercial conditions, from the point of view of animal welfare and meat quality, the most adequate pre-slaughter lairage time was 3 h for short travel. Pigs resting showed an increase in a relieve stress. Hence, holding pigs in lairage with toys for a few hours after arrival at the abattoir may at least improve mental state.8 pigs were slaughtered by directly killing heart and others 8 pigs were stunned by electricity. On arrival at the slaughterhouse, the pigs were slaughtered immediately. The results showed:①Electrical stun-kill pigs exhibited lower cortisol, ACTH, lactate when compared to directly killing group (P<0.05), Whereas no significant differences on LDH, CK and glucose were found (P>0.05).②The meat quality were not significantly affected by different slaughter measures (P>0.05).③Electrical stun-kill only decreased MCH (P<0.05), while others were not changed (P>0.05). It was concluded that pigs subjected to directly killing showed a more intense stress response than pigs subjected to electrical stun-kill. Accordingly, compared to those slaughter measures, we should preferentially choose electrical stun-kill methods, taking into account pig welfare.①Compared with the control group, cortisol in pig blood increased significantly higher about 40%(P<0.01). ACTH was increased about 15% and there was a significant difference (P<0.05).②Compared with the control group, only pH24 had significantly increased in stress group in the longissimus muscle (P<0.01). In the semimembranosus, meat quality including EC45min, EC24, pH45min, pH24 and MC45min in stress group were extremely significant (P<0.01) or significant level (P<0.05) when compared with control group. In addition, while only pH24 significant changes occurred in the longissimus muscle, other semimembranosus meat qualities in the stress and the control group had the same trend that occurred EC, pH had an upward trend, while MC had declining trend in the stress group compared with the control group.③There was a significantly notable positive correlation between cortisol and ACTH (r2= 0.225, P< 0.05); pH24 in Longissimus muscle and plasma cortisol levels showed significant positive correlation (r2=0.265, P<0.05), while had a negative correlation with ACTH (r2=-0.2195, P<0.05); EC45min, EC24, pH45min, pH24 and cortisol in the semimembranosus had significant positive correlations (P<0.05); At the same time, ACTH was also significantly positively related to MC24 (P<0.05); while EC45min was significantly negatively correlated with ACTH (P<0.05). Correlation between hormone stresses and the meat quality in the longissimus muscle and semimembranosus showed that the relevance of the positive and negative trend is essentially the same in both muscle tissues.④Cell stress model showed that:dexamethasone (DEX) had a significant cytotoxicity on muscle cells, resulting in a large number of intracellular LDH to escape into extracellular. Many nuclear bodies had become collapsed and then formed a clear DNA ladder, accompanies with marked change in cell form, then cells undergone cytoplasm damage and nuclear condensation damage. In addition, DEX forced a large number of ROS increased in the cytoplasm, resulting in oxidative stress. Flow tests further indicated that DEX could make the number of apoptosis and damage cells increased. The expression level of Bcl-2 was down-regulated while that of BAX was up-regulated, leading to increased ratio of pro-apoptotic BAX/Bcl-2 in a dose-dependent manner. The expression of Cytochrome C increased with the doses of DEX, and other apoptotic protein Caspase-3 expression had the same trend. The four apoptosis-related genes in gene and protein levels showed a similar trend. And both had a concentration-dependent effect. These effects could be prevented by isoconcentration of RU486. These results suggested that cortisol produced by pre-slaughter stress rapidly passed through the muscle cell membrane, disrupted normal physiological activities of cells. So that cells were oxidative stress, cell apoptosis immediately started and the process of apoptosis-related genes were activated, then cells showed significant apoptosis state. Abnormal muscle cells ultimately affected the meat quality after slaughter.The result showed:In the 11 pig tissues, GRa as well as the calcium ion channel-associated protein IP3R1 (Pumping the calcium ions from the sarcoplasmic reticulum into the cytoplasm) and SERCA1 (Pumping the calcium ions from the cytoplasm into the sarcoplasmic pump net) were both high expression in the longissimus muscle, which prompted that they may play a very important role in muscle tissue. In the C2C12 and pig primary muscle cells, DEX down-regulated GRa expression at the gene and protein levels, but can increase the number of GRa transposed to the nucleus. After DEX incubating, the two different calcium channels had different changes that IP3R1 expression increased as the concentration increased with DEX, while SERCA1 preached the opposite trend. As the muscle cells lacked capacity of pumping calcium into the sarcoplasmic reticulum, DEX resulted in calcium overload in the cytoplasm. The imbalance of calcium homeostasis leaded to activation of calcium-dependent protease and also activated apoptosis process, forcing cells from normal to apoptosis. This biological processes induced by DEX was almost completely prevented by co-incubation with RU486. A lot of literatures suggest that DEX the physiological role of DEX mediated by the nuclear regulatory factor GRa. In addition, Many GRa's nuclear regulatory functions were achieved by directly binding of GRa to GRE in the target gene transcriptional regulatory region. About 2.5 kb DNA fragment of pig IP3R1 and SERCA1 upstream of the transcription start sites were obtain using a Universal Genome-Walker method. Bioinformatics analysis showed that:327 bp before IP3R1 transcriptional start site had a high score of GRE components, while SERCA1 have none. About 1.5 kb deletion fragments before gene transcription start site were used to construct pGL3 reporter gene plasmid. When the deletion fragment missed GRE, the plasmid of IP3R1 promoter (pGL3-IP3R1-300) reduced its activity sharply. Compared with pGL3-IP3R1-300, pGL3-IP3R1-480 had a higher promoter activity, and which also had a trend of ascent as DEX concentration increased. The plasmid pGL-GRE specifically expressed GRE element, as well as RU486 could reduce the activity of plasmid containing the GRE. EMSA experiment confirmed that DEX-induced the expression of IP3R1 by GRαis dependent IP3R1 promoter region of the GRE element. The above results suggest that: stress increased cortisol, which translocated the active GRαinto the nucleus by membrane, and bound with GRE in the regulation region of IP3R1 to up-regulate the expression of IP3R1, so that calcium from the sarcoplasmic reticulum in a large number of lost to the cytoplasm, The imbalance of calcium homeostasis initiated apoptosis process, the damaged muscle eventually resulted in poor meat quality.Conclusion:①Considering the welfare, stress level and meat quality of pigs, choosing fasting 24 h, transport density below 275 kg/m2,3 h lairage with toys and electrical stun-kill measure were better.②Cortisol induced muscle cell apoptosis through binding cortisol to the hormone binding site in GRαand translocating the activated GRαinto nucleus. Then GRαbound with GRE in the regulation region of IP3R1 to up-regulate the expression of IP3R1, leading to cytoplasmic calcium overload and induced calcium signaling pathways mediated apoptosis, further affected the meat quality after slaughter.