The Mechanism Of Stunning Methods Affecting Meat Quality And Lipid Peroxidation Modulation In Broiler Chicks

The most widely used stunning methods (electrical stunning and gas stunning) were studied using 6 experiments. The effect of stunning methods on meat quality was systematically studied and nutritional modulation of lipid peroxidation was approached in broiler chicks by using the methods of absorption spectrometry, radiation immune assay, reversed-phase high-performance liquid chromatograph (RP-HPLC), electron spin resonance spectroscopy (ESR), real time quantitative RT-PCR (Reverse transcription-PCR). Firstly, the effects of different electrical and gas stunning parameters on meat quality were studied, and the parameters were optimized. Then, comparisons were done on the energy metabolism and lipid peroxidation in skeletal muscles between broilers stunned with electricity and gas mixtures. The possible mechanisms were further investigated through the studies on mitochondrial uncoupling proteins and MAPK-Nrf2/ECH-ARE signal transduction pathway. Eventually, Vitamin E (200 mg/kg DL-α- tocopherol) was supplemented in the diet to alleviate the oxidative damage in muscles from high-current-stunned broilers. The effect of VE on MAPK-Nrf2/ECH-ARE signal transduction pathway was also studied. Abstracts for experiments were listed as follows.Experiment 1 was designed to determine the effect of electrical stunning (ES) variables (low currents and high frequencies) on meat quality, glycolytic potential, and blood parameters in broilers. A total of 54 broilers were stunned with nine ES methods for 18 s, using sinusoidal alternating currents combining 35 V, 47 mA, 50 V, 67 mA, and 65 V, 86 mA with 160 Hz, 400 Hz, and 1,000 Hz. Samples for meat quality were obtained from the pectoralis major (PM) and musculus iliofibularis (MI), and samples for glycogen metabolism were taken from PM and tibialis anterior (TA) muscle at 45 min postmortem. The use of high frequency reduced the shear value in PM (400 Hz and 1,000 Hz vs. 160 Hz, P < 0.01), and cooking loss in MI (1,000 Hz vs. 160 Hz and 400 Hz, P < 0.01). The shear value of PM decreased at high frequency (400 Hz and 1,000 Hz) when current was high (50 V, 67 mA, and 65 V, 86 mA, P < 0.01), but increased at high frequency (1,000 Hz) when current was low (35 V, 47 mA). Stunning with 1,000 Hz (vs. 160 Hz) caused low glycogen, and glycolytic potential (GP) in PM (P < 0.05). Plasma corticosterone decreased (P < 0.05) at high currents (≥50 V, 67 mA), but was not affected by changes in frequency. Electrical current interacted with frequency in plasma glucose, a* 24h, shear value (PM), ultimate pH (MI) and GP (TA) (P < 0.05). This study indicated that high stunning frequencies (400 and 1,000 Hz) may potentially improve meat quality without aggregating stress when the current was not too low (more than 50 V, 67 mA).Experiment 2 was aimed to investigate the effects of different carbon dioxide (CO₂) concentrations on blood variables, glycolytic potential (GP), and meat quality of hot-boned muscles in broilers. Thirty broilers were exposed to one of the following five gas mixtures for 90 s: 40% CO₂ + 30% O₂ + N₂ (Control); 30% CO₂ +21%O₂ + N₂ (G30%); 40% CO₂ + 21% O₂ + N₂ (G40%); 50% CO₂ + 21% O₂ + N₂ (G50%); 60% CO₂ + 21% O₂ + N₂ (G60%). Samples were taken from the pectoralis major (PM), musculus iliofibularis (MI), and tibialis anterior muscles (TA) 45 min postmortem. The ultimate pH in both the PM (vs. G30% and G40%) and MI (vs. G40%, P < 0.05) was decreased with G60%, whereas drip loss in the PM (vs. G30%, P < 0.01) was increased with G60%. Drip loss in the MI (vs. control and G30%, P < 0.01) was increased with G50%. Lightness after 24 h (L*24h) in PM (vs. 30% and 40%, P < 0.01) was increased in group G50%. In MI muscle, L*24h was slightly decreased with G40% compared with the control (P < 0.10). The GP value in the PM was lower in the G30% and G40% than those in G60% (P < 0.05), and the GP value in the TA was the lowest in the G30% (P < 0.01). Plasma corticosterone, plasma glucose and meat quality (pH, redness, yellowness) 45 min postmortem were not affected by CO₂ levels (P > 0.05). In conclusion, stunning broilers with low CO₂ levels (30%, 40%) improved meat quality, but had no advantage in alleviating animal stress as compared with high CO₂ levels (50%, 60%).Experiment 3 was aimed to compare the effects of gas stunning (GS) and electrical stunning (ES) on energy metabolism in Arbor Acres broilers. Thirty-six birds were slaughtered without stunning (control) or after stunning with the following treatments: 40% CO₂ + 21% O₂ + N₂ (G40%);60% CO₂ + 21% O₂ + N₂ (G60%), 35 V, 47 mA, 400 Hz (E35V), 50 V, 67 mA, 160 Hz (E50V), and 65 V, 86 mA, 1,000 Hz (E65V). Muscle samples were obtained from the pectoralis major (PM) and tibialis anterior (TA) muscles 45 min postmortem and stored at ?80℃. Blood pH was decreased consistently with GS (G40% and G60%) compared with ES and the control (P < 0.01). No consistent differences were observed between GS and ES in the plasma variables, glycolytic potential (GP), adenosine phosphates, or fiber intensities. Plasma lactate was increased with G40% and E35V (P < 0.05), whereas plasma uric acid and urea nitrogen were increased with E35V (P < 0.05) as compared with the control. Compared with the control, the intensity of type IIB fibers decreased in broilers stunned with E35V and E50V (P < 0.05); and GP was increased (P < 0.01) with G60% in the breast muscle and reduced (P < 0.01) in the leg muscle with all the stunning treatments except for E50V. Energy was decreased (lower ATP, higher AMP and AMP to ATP ratio, P < 0.05) in breast muscle with G40% compared with ES at high currents (E50V and E65V). However, the adenosine phosphates with GS were not significantly different (P > 0.05) from ES at low current (E35V) either in breast or leg muscle. In conclusion, there was no essential difference in energy metabolism in broilers stunned with ES and GS, when ES was based on low current and high frequency and GS was based on hypercapnic hypercapnic moderate oxygenation stunning. The G40% is potentially a superior stunning variable among treatments from the view of energy metabolism.Experiment 4 was aimed to compare the effects of stunning methods (electrical stunning, ES vs. gas stunning, GS) on lipid peroxidation in broiler meat, and investigate possible mechanism through measuring plasma variables, muscle reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) concentrations, muscle fiber ratios, and mRNA levels of avian uncoupling protein (avUCP), avian adenine nucleotide translocator (avANT), and avian peroxisome proliferator-activated receptor-γcoactivator-1α(avPGC-1α). Arbor Acres broilers (AA, n = 36) were not stunned (control) or exposed to the following stunning treatments: 40% CO₂ + 21% O₂ + N₂ (G40%);60% CO₂ + 21% O₂ + N₂ (G60%); 35 V, 47 mA, 400 Hz (E35V), 50 V, 67 mA, 160 Hz (E50V), and 65 V, 86 mA, 1,000 Hz (E65V). The ROS level in tibialis anterior (TA) (P < 0.05) and the TBARS concentration in pectoralis major (PM) (P < 0.001) were decreased with GS groups compared with ES groups at 45 min postmortem. However, the TBARS concentrations 24 h postmortem in PM or TA were not affected by stunning methods (ES and GS). Compared with ES groups, the GS caused a higher expression of avUCP mRNA (1.47 fold in PM, and 2.41 fold in TA) and avPGC-1αmRNA (1.42 in PM and 2.08 fold in TA). In conclusion, the up-regulation of avUCP and avPGC-1αreduced the ROS accumulation and lipid peroxidation at 45 min postmortem in skeletal muscles of hypercapnic normal oxgenation gas stunned broilers. However, these changes were not sufficient to cause difference in the meat lipid peroxidation 24 h postmortem between hypercapnic moderate oxgenation GS and low current and high frequency ES.Experiment 5 was aimed to study the effect of stunning methods on meat color and lipid peroxidation, and explored the mechanism of peroxide scavenging through MAPK signaling pathway and its downstream SOD, GST expression in broilers. Totally 30 AA broilers were exposed to no stunning (control) or the following stunning treatments: 40% CO₂ + 21% O₂ + N₂ (G40%); G79% = 79% CO₂ + 21% O₂;E65V = 65 V,86 mA,1000 Hz;E150V =150 V,130 mA,60 Hz. Compared with the control, uric acid was increased in E65V, and meat discoloration was increased in G79% and E150V at 6 and 9 days postmortem(P < 0.05).Among the stunning treatments at 9 days postmortem, lightness (L*) was the lowest in G40%, and the highest in E150V (P < 0.01). In the breasts among the stunning treatments, the TBARS 45min, 3d, 6d, 9d levels were the highest with G40%, whereas the TBARS levels remained at low levels with E150V during the storage (P < 0.01). In the thigh, the TBARS 1d (P < 0. 10) and 6d (P < 0.01) concentrations were higher in G40% compared with E65V. In the thighs among the stunning groups, the activity of SOD 45min tended to be higher with G40% than that with E65V (P = 0.07), and the activity of GST 45min was higher than with the electrical stunning groups (P < 0.01); the activity of SOD 1d was increased in G40% compared with other stunning groups, and the activity of GST 1d was increased with G40% and E150V compared with E65V (P < 0.01). The activity of SOD 1d (P=0.03) and GST 1d (P=0.08) in thighs was increased respectively with G40% and G79% compared with the control.The G40% suppressed the transcriptional expressions of MAPK pathway (ERK1/2, JNK1, JNK2, p38) and its downstream antioxidant enzyme and Phase II enzymes (SOD1,SOD2,GSTA3,GSTK1,GSTM2,GSTT1) in breast . To the contrast, E65V up-regulated the expression of all the genes of MAPK pathway in breast and thigh muscles, and genes of antioxidant enzyme and Phase II enzymes excepted for SOD2 in thigh. Results showed that the MAPK-Nrf2/ECH-ARE pathway was suppressed by G40%, whereas activated by E65V.The level of G79% and E150V on MAPK pathway and antioxidant genes were among the treatments G40% and E150V. Overall, the MAPK pathway was mainly suppressed by GS (especially low CO₂ concentration), whereas, mainly activated by ES (especially low current + high frequency). The meat color was well maintained by both G40% and E65V. The E65V increased serum uric acid, activated the expression of MAPK-Nrf2/ECH-ARE pathway genes and its downstream SOD and GST genes, showing a good adaptation reaction to slaughter stress, thus caused an increase in the antioxidant ability and delayed meat lipid peroxidation thereafter. However, G40% suppressed the antioxidant genes and MAPK pathway, showing a poor adaptation reaction, and led to higher meat oxidation during prolonged meat storage.Experiment 6 was aimed to study the effect of dietary VE on meat lipid peroxidation induced by high current/low frequency electrical stunning (ES) in broilers, and explored the molecular mechanism through MAPK-Nrf2/ECH-ARE signaling pathway. Totally 240 1-day-old AA broilers were distributed to 4 treatments, with 6 replicates in each treatment, and 10 broilers per replicate. Broilers were fed with normal diet (20 mg/kg DL-α- tocopherol, VEa) in the first stage (days 0～17). A basal diet (no supplementation ofα- tocopherol, VEb) and treatment diet (200 mg/kg DL-α- tocopherol, VEc) were prepared for the second stage (days 18～38). The control birds were fed with VEb through days 18～38; Birds from treatments VE1w, VE2w, VE3w were firstly fed with VEb, and then with the diet VEc (200 mg/kgα- tocopherol) respectively since day 32, 25, and 18. On day 38, one bird per replicate was selected (body weight 2.0±0.2), stunned with an alternative current of 150V, 130 mA, 60Hz, for 1s. Pectoralis major (PM, breast) and musculus iliofibularis (MI, thigh) were taken for measurements. TBARS concentrations were decreased in breast (45min, 2d, 6d postmortem) and thigh (45min, 2d, 4d, 6d postmortem) muscles with VE groups (VE1w, VE2w, VE3w, P < 0.01). The TBARS concentration order was that control > VE3w > VE2w > VE1w in thigh at 2d postmortem (P < 0.01). Compared with the control, the precipitation of VE was increased in breast and thigh muscles with VE supplementation groups, with the order of VE1w > VE2w > VE3w > control (P < 0.01). Compared with the control in breast, the gene expressions of JNK1, JNK2 and ECH (chicken Nrf2) in the MAPK-Nrf2/ECH-ARE pathway was decreased by VE1w and VE2w, whereas the expression of JNK1, JNK2 and ECH was increased in VE3w. Compared with the control in thigh, JNK1, JNK2, p38 and ECH were down-regulated by VE1w, while up-regulated by VE2w. The JNK1 and ECH were up-regulated by VE3w in thigh. The activity of SOD and GST in breast was not affected by VE supplementation (P > 0.05). Compared with the thigh of control, activity of GST 45min was decreased with VE1w (P < 0.01), and the activity of GST 2d was increased with VE1w and VE2w (P < 0.01). Conclusion, a VE supplementation for 1 week caused the highest VE level in muscles, which decreased the slaughter stress, suppressed the MAPK-Nrf2/ECH-ARE oxidative stress signaling, alleviated the oxidative stress and meat oxidation in broilers.