高温对肉鸡胸肌卫星细胞肌浆网钙调节及肉品质的影响
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摘要
现代肉用家禽生产过程中,肉品质下降已经成为人们关注的一个焦点问题。环境高温导致肌肉pH值下降、肉色苍白以及系水力下降等。而目前关于环境高温是否通过升高钙离子浓度,进而导致家禽肉品质下降尚不明确。因此,通过动物试验和体外细胞培养试验研究高温对肉鸡肉品质的影响及其机理,得出:
1、日循环高温对肉鸡直肠温度、呼吸率、丙二醛含量及抗氧化酶活性的影响
日循环高温升高肉鸡呼吸率和直肠温度(P<0.05),并且增强肝脏抗氧化酶SOD以及GSH-PX的活性,升高MDA含量(P<0.05);此外,日粮中添加150mg/kg的维生素E能够降低肝脏MDA含量(P<0.05),并有升高肝脏抗氧化酶活性的趋势(P>0.05)。
2、日循环高温对肉鸡胸肌乳酸产生量及肉品质的影响
在人工环境控制舱内模拟日循环高温,发现持续7天以及14天的日循环高温应激(27-35-27℃),增加肉鸡胸肌乳酸的产生量(P<0.05),降低宰后胸肌pH值(P<0.05),使肌肉L值升高(P<0.05)肉色苍白,胸肌剪切力和滴水损失升高(P<0.05);而日粮中补充维生素E,降低宰后胸肌剪切力和滴水损失(P<0.05),减慢pHi以及pHu的下降速度(P<0.05),但对肉色的L值无明显改变(P>0.05),并有降低胸肌乳酸产生量的趋势,但差异不显著(P>0.05)。
3、高温对肉鸡胸肌卫星细胞胞浆钙离子浓度及乳酸产生量的影响
肉鸡胸肌卫星细胞在25℃以及40℃恒温处理30min过程中钙离子浓度变化无显著差异(P>0.05);而高温应激(44℃)可显著升高肉鸡胸肌卫星细胞胞浆钙离子浓度(P<0.05);并且引起卫星细胞内乳酸产生量呈上升趋势,且在高温处理50min时显著升高(P<0.05)。
4、肉鸡胸肌卫星细胞胞浆钙离子对乳酸产生量的影响
肉鸡胸肌卫星细胞高温(44℃)条件下恒温处理1小时细胞内的乳酸含量比适温(40℃)恒温处理1小时显著升高(P<0.05);当在适温条件下添加2mM的钙离子载体后,细胞内游离钙离子浓度升高,卫星细胞乳酸产生量升高,但未达到差异显著水平(P>0.05);卫星细胞在高温(44℃)条件下恒温处理1小时过程中加入细胞内钙螯合剂(BAPTA-AM)后,细胞内游离钙离子浓度降低,细胞内乳酸含量降低,且与对照组及第四组(高温处理组)相比差异显著(P<0.05);试验证明:环境高温下肉鸡胸肌卫星细胞通过升高胞浆钙离子浓度,增加细胞内乳酸的产生量。
5、高温对肉鸡卫星细胞肌浆网RyR及InsP3受体的影响
通过高温条件下添加肌浆网受体抑制剂研究发现,高温通过激活肉鸡胸肌卫星细胞肌浆网钙释放通道上的RyR受体,释放肌浆网中钙离子到胞浆,进而升高胞浆钙离子浓度(P<0.05);而高温条件下InsP3受体活性变化差异不显著(P>0.05),对胞浆钙离子浓度的改变无显著作用。
综上得出:环境高温通过激活肉鸡胸肌卫星细胞肌浆网上钙释放通道的RyR受体,升高胞浆钙离子浓度,引起乳酸产生量增加,导致胸肌pH值下降,进而导致肉色苍白、滴水损失增加、肉品质下降。
In modern society, Inferior meat quality has been a great increasing problem which people pay more attention. As already known heat temperature is capable of causing lower pH、pale meat and a reduction in water-holding capacity, and so on. Whereas high temperature impact the quality of poultry meat via increasing Ca2+ concentration is not clear. Therefore, In this study, we will sought to determine the mechanism in vivo and in vitro initially.
1、Effect of high temperature on rectal temperature, respiration rate and MDA, SOD,GSH-PX of broilers pectoral muscle
Three hundreds male broilers were divided into four groups with eight replicates, fifteen broilers per replicate. Group one was control group, kept in 25℃, group two was pari-feeding group with the temperature of 25℃, group three was a high-temperature group, and group was high temperature +vitamin E. Broilers were killed at the seventh day and the fourteenth day.
The ambient high temperature leaded to increase the rectal temperature and respiration rate significantly(P<0.05); this also increased the activity of MDA, SOD and GSH-PX(P<0.05). Furthermore, addition of vitamin E(150mg/kg) tend to decrease the rectal temperature, and respiration rate, but kept non-significantly.
2、Effect of high temperature on lactate content and the meat quality of broilers pectoral muscle
In environment control chambers, broilers were kept for 7days and 14days. The experiment proved that at both the 7th day and 14th day high temperature lead to an increasing of lactate content(P<0.05), then pHi and pHu decreased(P<0.05). And L*,drip loss and shear force increased in pectoral muscle was found. Addition vitamin E in diet, tend to decrease the lactate content, but non-significantly(P<0.05). Whereas decreased the drip loss, shear force,significantly, caused an increase of pHi and pHu(P<0.05) .
3、Impact of the ambient high temperature on cytosolic Ca2+concentration and lactate levels in satellite cells in the pectoral muscle of broilers
Primary satellite cells were cultured in 96-well plates. Experiment consisted of 2 treatment groups: a high-temperature group (treated at 40℃,then to 44℃30 minutes later) and a control group (at 25℃). Eight wells were used for each group and 100μL (1×106cells/mL) of suspended satellite cells was added to each well. Then the Ca2+concentration was recorded fluorescence microplate reader.At 25℃and 40℃the cytosolic calcium concentration remained almost stable, and kept non-significantly(P>0.05). At 44℃however, the cytosolic calcium concentration increased significantly with the 30 minutes(P<0.05).
six groups of primary satellite cells from the breast tissue of broilers were treated separately at a high temperature(44℃) for 10, 20, 30, 40, 50 and 60min. The cytosolic lactate content was then determined using commercial colorimetric diagnostic kits from the Jiancheng Biochemical Institute. The cytosolic lactate in primary satellite cell of breast tissue from broilers tended to increase over the period of 1 hr. When the suspended cells were cultured from 40 to 50min at 44℃, the cytosolic lactate content increased significantly(P<0.05).
The experiment proved that the cyclic high temperature increase the cytosolic calcium concentration and lactate content.
4、Impact of cytosolic calcium concentration to lactate production
The experimental samples were allocated to 5 treatment groups: Group 1 was a control group containing only suspended satellite cells, Group 2 contained suspended satellite cells and 2mM DMSO, and Group 3 contained suspended satellite cells and 2mM calcium ionophore(A23187), these three groups were all incubated at 40℃for an hour. Group 4 was a high-temperature group contained the same concentration of suspended cells, and Group5 with the addition of 1mM intracellular Ca2+ chelating agent(BAPTA-AM). These two groups were cultured at 44℃for 1 hr.
Under these conditions, the intracellular Ca2+ concentration in Group 2 and 3 increased and was coupled with a non-significantly increase in lactate content (P>0.05). Group 4 was cultured in 44℃, and the lactate content was significantly(P<0.05) higher then that of Group 1 which was cultured at 40℃. In group 5 injected which was treated with BAPTA-AM, the intracellular Ca2+ concentration decreased and the lactate content decreased significantly(P<0.05). The experiment proved that altering the intracellular Ca2+ concentration impacts on the cytosolic lactate content. High temperature impact on cytosolic lactate content of sallitate cells by changing the intracellular Ca2+ concentration.
5、Impact of high temperature on the activity of RyR and InsP3 receptor in satellite cells
The experimental samples were allocated to 3 treatment groups: Group 1 was a control group containing only suspended satellite cells, Group 2 contained suspended satellite cells and 50μmol/L ruthenium red, and Group 3 contained suspended satellite cells and 50μmol/L 2-Aminoethoxydiphenyl borate (2-APB). Three groups were all cultured at 44℃for an hour.
The cytosolic Ca2+ concentration in group 1 , group 2 and group 3 tend to increased, but group 2 kept significantly lower then group 1 (P<0.05). Group 3 and group 1 has non-significantly difference(P>0.05). The experiment proved that the ambient high temperature activated the RyR calcium channel, and released calcium to cytosolic, then increased the intracellular Ca2+ concentration; whereas the InsP3 calcium channel has non-siginificant effect in the process.
In summary, the present finding that high temperature conditions increase the intracellular Ca2+ concentration and lactate formation via activity the RyR receptor on sarcoplasmic reticulum of the satellite cells. And then, lead to a lower pH、paler meat、higher drip loss and final in inferior meat quality.
1、日循环高温对肉鸡直肠温度、呼吸率、丙二醛含量及抗氧化酶活性的影响
日循环高温升高肉鸡呼吸率和直肠温度(P<0.05),并且增强肝脏抗氧化酶SOD以及GSH-PX的活性,升高MDA含量(P<0.05);此外,日粮中添加150mg/kg的维生素E能够降低肝脏MDA含量(P<0.05),并有升高肝脏抗氧化酶活性的趋势(P>0.05)。
2、日循环高温对肉鸡胸肌乳酸产生量及肉品质的影响
在人工环境控制舱内模拟日循环高温,发现持续7天以及14天的日循环高温应激(27-35-27℃),增加肉鸡胸肌乳酸的产生量(P<0.05),降低宰后胸肌pH值(P<0.05),使肌肉L值升高(P<0.05)肉色苍白,胸肌剪切力和滴水损失升高(P<0.05);而日粮中补充维生素E,降低宰后胸肌剪切力和滴水损失(P<0.05),减慢pHi以及pHu的下降速度(P<0.05),但对肉色的L值无明显改变(P>0.05),并有降低胸肌乳酸产生量的趋势,但差异不显著(P>0.05)。
3、高温对肉鸡胸肌卫星细胞胞浆钙离子浓度及乳酸产生量的影响
肉鸡胸肌卫星细胞在25℃以及40℃恒温处理30min过程中钙离子浓度变化无显著差异(P>0.05);而高温应激(44℃)可显著升高肉鸡胸肌卫星细胞胞浆钙离子浓度(P<0.05);并且引起卫星细胞内乳酸产生量呈上升趋势,且在高温处理50min时显著升高(P<0.05)。
4、肉鸡胸肌卫星细胞胞浆钙离子对乳酸产生量的影响
肉鸡胸肌卫星细胞高温(44℃)条件下恒温处理1小时细胞内的乳酸含量比适温(40℃)恒温处理1小时显著升高(P<0.05);当在适温条件下添加2mM的钙离子载体后,细胞内游离钙离子浓度升高,卫星细胞乳酸产生量升高,但未达到差异显著水平(P>0.05);卫星细胞在高温(44℃)条件下恒温处理1小时过程中加入细胞内钙螯合剂(BAPTA-AM)后,细胞内游离钙离子浓度降低,细胞内乳酸含量降低,且与对照组及第四组(高温处理组)相比差异显著(P<0.05);试验证明:环境高温下肉鸡胸肌卫星细胞通过升高胞浆钙离子浓度,增加细胞内乳酸的产生量。
5、高温对肉鸡卫星细胞肌浆网RyR及InsP3受体的影响
通过高温条件下添加肌浆网受体抑制剂研究发现,高温通过激活肉鸡胸肌卫星细胞肌浆网钙释放通道上的RyR受体,释放肌浆网中钙离子到胞浆,进而升高胞浆钙离子浓度(P<0.05);而高温条件下InsP3受体活性变化差异不显著(P>0.05),对胞浆钙离子浓度的改变无显著作用。
综上得出:环境高温通过激活肉鸡胸肌卫星细胞肌浆网上钙释放通道的RyR受体,升高胞浆钙离子浓度,引起乳酸产生量增加,导致胸肌pH值下降,进而导致肉色苍白、滴水损失增加、肉品质下降。
In modern society, Inferior meat quality has been a great increasing problem which people pay more attention. As already known heat temperature is capable of causing lower pH、pale meat and a reduction in water-holding capacity, and so on. Whereas high temperature impact the quality of poultry meat via increasing Ca2+ concentration is not clear. Therefore, In this study, we will sought to determine the mechanism in vivo and in vitro initially.
1、Effect of high temperature on rectal temperature, respiration rate and MDA, SOD,GSH-PX of broilers pectoral muscle
Three hundreds male broilers were divided into four groups with eight replicates, fifteen broilers per replicate. Group one was control group, kept in 25℃, group two was pari-feeding group with the temperature of 25℃, group three was a high-temperature group, and group was high temperature +vitamin E. Broilers were killed at the seventh day and the fourteenth day.
The ambient high temperature leaded to increase the rectal temperature and respiration rate significantly(P<0.05); this also increased the activity of MDA, SOD and GSH-PX(P<0.05). Furthermore, addition of vitamin E(150mg/kg) tend to decrease the rectal temperature, and respiration rate, but kept non-significantly.
2、Effect of high temperature on lactate content and the meat quality of broilers pectoral muscle
In environment control chambers, broilers were kept for 7days and 14days. The experiment proved that at both the 7th day and 14th day high temperature lead to an increasing of lactate content(P<0.05), then pHi and pHu decreased(P<0.05). And L*,drip loss and shear force increased in pectoral muscle was found. Addition vitamin E in diet, tend to decrease the lactate content, but non-significantly(P<0.05). Whereas decreased the drip loss, shear force,significantly, caused an increase of pHi and pHu(P<0.05) .
3、Impact of the ambient high temperature on cytosolic Ca2+concentration and lactate levels in satellite cells in the pectoral muscle of broilers
Primary satellite cells were cultured in 96-well plates. Experiment consisted of 2 treatment groups: a high-temperature group (treated at 40℃,then to 44℃30 minutes later) and a control group (at 25℃). Eight wells were used for each group and 100μL (1×106cells/mL) of suspended satellite cells was added to each well. Then the Ca2+concentration was recorded fluorescence microplate reader.At 25℃and 40℃the cytosolic calcium concentration remained almost stable, and kept non-significantly(P>0.05). At 44℃however, the cytosolic calcium concentration increased significantly with the 30 minutes(P<0.05).
six groups of primary satellite cells from the breast tissue of broilers were treated separately at a high temperature(44℃) for 10, 20, 30, 40, 50 and 60min. The cytosolic lactate content was then determined using commercial colorimetric diagnostic kits from the Jiancheng Biochemical Institute. The cytosolic lactate in primary satellite cell of breast tissue from broilers tended to increase over the period of 1 hr. When the suspended cells were cultured from 40 to 50min at 44℃, the cytosolic lactate content increased significantly(P<0.05).
The experiment proved that the cyclic high temperature increase the cytosolic calcium concentration and lactate content.
4、Impact of cytosolic calcium concentration to lactate production
The experimental samples were allocated to 5 treatment groups: Group 1 was a control group containing only suspended satellite cells, Group 2 contained suspended satellite cells and 2mM DMSO, and Group 3 contained suspended satellite cells and 2mM calcium ionophore(A23187), these three groups were all incubated at 40℃for an hour. Group 4 was a high-temperature group contained the same concentration of suspended cells, and Group5 with the addition of 1mM intracellular Ca2+ chelating agent(BAPTA-AM). These two groups were cultured at 44℃for 1 hr.
Under these conditions, the intracellular Ca2+ concentration in Group 2 and 3 increased and was coupled with a non-significantly increase in lactate content (P>0.05). Group 4 was cultured in 44℃, and the lactate content was significantly(P<0.05) higher then that of Group 1 which was cultured at 40℃. In group 5 injected which was treated with BAPTA-AM, the intracellular Ca2+ concentration decreased and the lactate content decreased significantly(P<0.05). The experiment proved that altering the intracellular Ca2+ concentration impacts on the cytosolic lactate content. High temperature impact on cytosolic lactate content of sallitate cells by changing the intracellular Ca2+ concentration.
5、Impact of high temperature on the activity of RyR and InsP3 receptor in satellite cells
The experimental samples were allocated to 3 treatment groups: Group 1 was a control group containing only suspended satellite cells, Group 2 contained suspended satellite cells and 50μmol/L ruthenium red, and Group 3 contained suspended satellite cells and 50μmol/L 2-Aminoethoxydiphenyl borate (2-APB). Three groups were all cultured at 44℃for an hour.
The cytosolic Ca2+ concentration in group 1 , group 2 and group 3 tend to increased, but group 2 kept significantly lower then group 1 (P<0.05). Group 3 and group 1 has non-significantly difference(P>0.05). The experiment proved that the ambient high temperature activated the RyR calcium channel, and released calcium to cytosolic, then increased the intracellular Ca2+ concentration; whereas the InsP3 calcium channel has non-siginificant effect in the process.
In summary, the present finding that high temperature conditions increase the intracellular Ca2+ concentration and lactate formation via activity the RyR receptor on sarcoplasmic reticulum of the satellite cells. And then, lead to a lower pH、paler meat、higher drip loss and final in inferior meat quality.
引文
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