【作者】 唐仁勇；

【导师】 陈代文；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2008， 博士

【摘要】 在肉的食用品质中,肉的嫩度是消费者最为关心和重视的性状之一。研究显示μ-Calpain和Calpastatin两种蛋白与肉的嫩化密切相关。为了初步探索μ-Calpain和Calpastatin基因表达与猪肉品质的关系,营养因素对猪肉品质(尤其是嫩度)以及对μ-Calpain和Calpastatin基因表达的作用效果及其程度,本研究在建立猪肉嫩度与μ-Calpain和Calpastatin基因表达的相关关系基础上,考察了营养水平和MgAsp对猪μ-Calpain和Calpastatin基因表达的影响,初步探明μ-Calpain和Calpastatin基因表达的营养调控效果,并进一步从分子水平上研究了镁对μ-Calpain、Calpastatin基因表达的调控。本研究对于深入认识猪肉肉质性状形成原理及营养与肉质的关系具有理论意义,对进一步研发改善猪肉品质的营养技术、促进养猪生产具有重要指导意义。本研究包括以下六个试验:试验一:考察不同品种猪肌肉中μ-Calpain和Calpastatin mRNA的表达差异,并与猪肉嫩度之间建立相关关系。选体重在80-90kg的长白猪(6头)和太湖猪(6头)进行屠宰。屠宰时取相同部位的骨骼肌(眼肌)用于检测肉的嫩度及μ-Calpain和Calpastatin mRNA水平。结果表明:与长白猪相比,太湖猪眼肌具有更低的剪切力(p＜0.05),而其μ-Calpain mRNA水平极显著高于长白猪,相关性分析发现肌肉中μ-Calpain mRNA水平与剪切力呈显著的负相关。试验二:考察营养水平对D×L×Y育肥猪肉质性状及μ-Calpain、Calpastatin基因表达的影响。采用单因子试验设计,将24头健康的D×L×Y杂交猪(起始重64kg左右)随机分到2个处理,每个处理3个重复,每个重复4头猪。两个处理分别饲喂高、低营养水平(DE分别为13.81,12.55 MJ/kg;CP分别为14,11%)的饲粮。当体重达到100kg左右时,每个处理选6头猪进行屠宰取样。测定猪的常规肉质性状指标,并用实时荧光定量PCR方法检测骨骼肌μ-Calpain、Calpastatin基因mRNA的表达量。结果表明:饲喂低营养水平日粮(DE=12.55MJ/kg,CP=11.08%)增加了眼肌肌内脂肪含量,降低了眼肌的剪切力(p＜0.05),并提高了μ-Calpain mRNA表达量(p＜0.05)。说明营养水平可以影响猪肉品质和μ-Calpain mRNA的表达。试验三:考察日粮短期添加MgAsp对试猪血液理化指标、猪肉品质以及眼肌μ-Calpain和Calpastatin mRNA含量的影响。试验采用2×2因子试验设计。将24头健康的D×L×Y杂交猪(起始重90kg左右)随机分到对照和加镁(1000mg Mg/kg)处理。饲喂处理日粮9d后,在两处理中随机选6头试猪实施运输应激(1.5h),宰前静脉采血,屠宰所有试猪。测定试猪血液理化指标、常规肉质性状指标,并用实时荧光定量PCR方法检测眼肌μ-Calpain、Calpastatin基因mRNA的表达量。结果表明:运输应激增加了血清中钙(p＜0.05)、磷(p＜0.001)、葡萄糖(p＜0.001)和皮质醇水平(p＜0.01),同时镁(p=0.078)也有所增加。日粮添加MgAsp增加了血清镁的含量(p=0.057)。运输应激导致宰后45min(p＜0.05)和24h(p＜0.05)的眼肌L值都有所下降,并降低了眼肌45minb值(p=0.073),且增加了臀肌45min pH(p＜0.05)和眼肌45min pH(p=0.098),但运输使臀肌72h(p＜0.05)、眼肌24h(p＜0.01)和72h(p＜0.01)的剪切力有所提高。添加MgAsp后降低了臀肌45min(p＜0.05)和24h(p＜0.05)的L值,加镁也降低了眼肌宰后45min的L值(p＜0.05),并增加了臀肌45min时的a值(p＜0.05),在数值上加镁有降低剪切力的趋势,但对pH值和滴水损失没有影响。运输应激增加了Calpastatin基因的表达(p＜0.05),而添加MgAsp有提高μ-Calpain表达量的趋势(p=0.079)。这些结果表明,运输应激(1.5h)提高宰后猪肉的肉色和pH值,降低嫩度;MgAsp对肉色有改善作用,有降低剪切力的趋势,但对pH值和滴水损失没有明显影响。试验四:在试验三的基础上,通过添加两个水平的MgAsp以及在宰前对猪只实施运输应激,考察镁缓解应激及各处理对猪血液、组织生化指标、肉品质和肌肉中μ-Calpin、Calpastatin mRNA含量的影响程度。选用体重约88kg D×L×Y杂交猪36头,分为三组,在宰前5d分别饲喂添加不同镁水平的日粮,处理1、2和3分别为基础、1000mg Mg/kg和2000mg Mg/kg。每组选取一半猪只对其实施2h运输应激,之后立即将所有试猪屠宰。检测试猪血液、组织理化指标、猪肉品质以及用实时荧光定量PCR测定试猪眼肌μ-Calpain和Calpastatin基因mRNA含量。结果表明:运输应激增加了血清中钙(p＜0.01)、葡萄糖(p＜0.01)和皮质醇(p＜0.01)的水平,同时肌肉中cAMP(p=0.09)也有所增加,运输降低了肌肉中糖原含量(p＜0.05)。日粮添加MgAsp增加了血清镁(p＜0.05),肌肉中糖原(p＜0.05)的含量,降低了血清中NOS活性(p＜0.05)。运输应激导致眼肌24h(p＜0.05)、眼肌72h(p＜0.05)和臀肌24、72h(p＜0.05)的剪切力都有所增加,从而降低了肉的嫩度。运输使眼肌宰后45min pH(p＜0.01)和24h pH(p＜0.05)下降,并极显著降低了臀肌宰后45min pH。添加MgAsp有降低剪切力的趋势,在实施运输应激后,加镁可显著降低剪切力。添加MgAsp对肌肉pH基本无影响,可显著降低肌肉的滴水损失,显著增加眼肌宰后45min的a值,对眼肌宰后24h的a值也有增加的趋势;并降低了眼肌24h(p=0.05)和臀肌24h(p=0.05)的L值。运输增加了Calpastatin基因的表达(p=0.05),添加MgAsp有提高μ-Calpain表达量的趋势。这些结果表明,运输应激(2h)降低部分肉质;MgAsp对肉色和滴水损失有改善作用,有降低剪切力的趋势,但对pH值没有影响。试验五:利用实时定量RT-PCR、SDS-PAGE等方法研究镁离子、肾上腺素对大鼠L6成肌细胞中μ-Calpain和Calpastatin mRNA水平以及对L6细胞内蛋白降解程度的影响。采用单因素试验设计,试验共7个处理:1-对照组;2-0.5 mM Mg组;3-1mMMg组;4-0.5 mM Mg+100ng/ml肾上腺素组;5-50ng/ml肾上腺素组;6-100ng/ml肾上腺素组;7-200ng/ml肾上腺素组。结果表明:与对照组相比,添加两种剂量的镁离子均显著增加成肌细胞μ-Calpain mRNA水平。此外,1mM镁离子组还显著增加Calpastatin mRNA水平。与对照组相比,添加肾上腺素都显著或极显著地增加μ-Calpain和Calpastatin mRNA水平,200ng/ml肾上腺素组的Calpastatin mRNA水平显著高于肾上腺素50ng/ml组,而μ-Calpain mRNA水平在各肾上腺素处理组没有显著差异。此外,0.5mM镁离子与100ng/ml肾上腺素联合添加组显著增加μ-Calpain mRNA水平,对Calpastatin mRNA水平无影响。添加肾上腺素极显著地增加细胞内cAMP的浓度,肾上腺素100ng/ml组的cAMP浓度显著高于肾上腺素50ng/ml组。处理3组和处理7组细胞内蛋白降解较为明显,而其余处理组蛋白降解无较大差异。试验六:本实验拟通过体外培养大鼠骨骼肌成肌细胞,在添加镁离子的同时,加入μ-Calpain抑制剂-E64,在定量检测μ-Calpain基因mRNA水平基础上进一步检测μ-Calpain及其底物蛋白Nebulin亚片段表达量的变化。从而进一步研究镁离子与μ-Calpain的表达、肌肉蛋白降解以及与肉品质的关系。试验设4个处理:1-对照组;2-加镁组;3-E64组;4-加镁+E64组。其中镁的处理浓度为1.0mM.;E64的处理浓度为10μM。结果表明:培养液中添加1mM镁离子可显著增加μ-Calpain mRNA水平,并显著增加μ-Calpain的蛋白量,并有增加Nebulin蛋白200 kDa亚片段的趋势(p=0.096);培养液中添加E64极显著地降低Nebulin蛋白200 kDa亚片段的量。综上所述,本研究结果表明:1)μ-Calpain、Calpastatin基因与猪肉嫩度密切相关。μ-Calpain基因表达增加提高猪肉嫩度:Calpastatin基因表达增加降低猪肉嫩度。2)μ-Calpain、Calpastatin基因表达受营养水平、镁和应激的调控:低营养水平和添加镁可以促进μ-Calpain的表达,并促进猪肉嫩度的提高;应激显著增加Calpastatin的表达,并导致猪肉嫩度下降。3)由本试验结果可初步证明,营养水平、镁及应激对μ-Calpain、Calpastatin基因表达的调控效果是这些因素影响猪肉嫩度的机制之一。更多还原

【Abstract】 Tenderness is one of the most important factors influencing the acceptability of meat.It is well established thatμ-calpain and calpastatin have a strong relation with meat tenderness.The objective of this study was to investigate the effects of nutrients in swine finishing diets on pork quality(especially for tenderness)and the mRNA abundance ofμ-calpain and calpastatin of muscle in finishing pigs and relative mechanism.The correlation between the Warner-Bratzler shear force(WBSF)and the mRNA abundance ofμ-calpain and calpastatin was firstly developed,then determined the effects of the different nutritional level and supplementing swine finishing diets with magnesium aspartate (MgAsp)on pork quality and the mRNA abundance ofμ-calpain and calpastatin of muscle in finishing pigs,and then investigated how the supplemental magnesium affected the tenderness in vitro.This study is of both academic and practical value to further understand the development of pork quality at molecular level and the interaction between pork quality and nutrition.Our studies included six experiments.Experiment 1:Six Landrace pigs and six Taihu pigs with 80-90 kg BW were slaughtered to investigate the difference of tenderness and gene expression ofμ-calpain and calpastatin of longissimus muscle(LM).The results indicated that Taihu pigs had lower WBSF(p＜0.05)and higherμ-calpain mRNA abundance(p＜0.01)of longissimus muscle(LM)compared with Landrace pigs,and the abundance ofμ-calpain mRNA had a negative correlation(P＜0.05)with the WBSF of LM.Experiment 2:To determine effects of nutritive levels(nutrient density)on meat quality and gene expression ofμ-calpain and calpastatin of LM in DLY(Duroc×Large White×Yorkshire)finishing pigs,24 DLY pigs with～64kg BW were allotted randomly to feed either high(DE=13.81MJ/kg,CP=14.19%)or low(DE=12.55MJ/kg,CP=11.08%) nutritive level.Six pigs were slaughtered in each group when pigs reached 100kg BW.The results indicated that the pigs fed low nutritive level had more intramuscular fat content, lower WBSF(p＜0.05)and higher abundance ofμ-calpain mRNA(p＜0.05)in LM than the pigs fed high level.It is concluded thatμ-calpain gene expression and pork quality may be affected by dietary nutritional level. Experiment 3:To investigate effects of supplemental magnesium aspartate(MgAsp)on blood parameters,pork quality and gene expression ofμ-calpain and calpastain in muscle of finishing pig.Twenty-four crossbred DLY finishing pigs(mean live weight of 90 kg) were assigned randomly to 0 and 1000 mg of supplemental Mg from MgAsp per kg of diet for 9 d before slaughter.Then 6 pigs from each dietary treatment were selected randomly and subjected either to no transportation stress(NTS)or 1.5 h of transportation stress(TS). The results indicated that transportation increased serum concentrations of Ca(p＜0.05),P (p＜0.001),glucose(p＜0.001)and cortisol(p＜0.01).Supplementation of MgAsp increased concentration of serum Mg(p=0.057).Transportation decreased L~* value of LM(p＜0.05)at 45 min and 24 h,and b~* value of LM(p=0.073)at 45 min after slaughter.Furthermore,transportation increased pH value of biceps femoris(BF)at 45 min (p＜0.05)and LM at 45 min(p=0.098)after slaughter,respectively.Transportation increased WBSF value of BF at aging 72 h(p＜0.05)and LM(p＜0.01)at aging 24h and 72h,respectively.Pigs fed the MgAsp-supplemented diet had lower L~* value(p＜0.05)of LM at 45 min,and BF at 45 min and at 24 h after slaughter than pigs fed with the control diet.Supplementation of MgAsp increased a~* value of BF at 45 min(p＜0.05)and decreased WBSF value(p＜0.05).Transportation improved mRNA level of calpastatin of muscle(p＜0.05).Meanwhile,supplementation of MgAsp increased mRNA level ofμ-calpain of muscle(p=0.079).These results suggested that transportation(1.5h)stress increased postmortem color and pH value of pork and decreased tenderness of pork,and supplemental MgAsp improved color of pork and had a trend in declining WBSF,but did not influence drip loss and pH value of pork.Experiment 4:The objective of this study was to investigate the effects of supplementing swine finishing diets with two levels of MgAsp and short-duration transportation stress on blood parameters,pork quality and the mRNA abundance ofμ-calpain and calpastatin of muscle in finishing pigs.Thirty-six crossbred finishing pigs (～88 kg BW)were assigned randomly to 0,1000,or 2000 mg of supplemental Mg from MgAsp per kg of diet for 5 d before slanghter.Then 6 pigs from each dietary treatment were selected randomly and subjected either to NTS or 2 h of TS.Transportation stress resulted in the higher concentrations(p＜0.01)of serum Ca,glucose and cortisol,lower pH(p＜0.01),higher WBSF(p＜0.05),higher cAMP concentration and higher calpastatin mRNA abundance(p=0.05)of LM compared with NTS treatments.Supplementation of MgAsp in TS treatments improved serum Mg concentrations(p＜0.05),glycogen in LM, decreased the activity of NOS in serum,reduced drip loss percent(p＜0.05)and 24-h L~* value(p＜0.05)of LM and BF and increased 45-min a~* value of LM(p＜0.05),and decreased WBSF(p＜0.05)compared to TS treatments fed the control diet.Moreover, supplementation of MgAsp had a trend to increaseμ-calpain mRNA abundance(p=0.13) of muscle.It is concluded that supplementation of MgAsp improves water-holding capacity and pork color,and alleviates the negative effects of transportation stress on meat tenderness.Experiment 5:The rat L6 myoblast was used to investigate effect of the magnesium and epirenamine supplemented on the gene expression ofμ-calpain and calpastatin.The experiment had seven treatments:control,0.5 mM,1 mM of supplemental Mg from MgCl, 0.5 mM Mg+epirenamine at 100ng/ml concentration,50ng/ml,100ng/ml and 200 ng/ml epirenamine.The results indicated that supplementation of Mg improved significantly the abundance ofμ-calpain mRNA at both two doses of Mg,and increased calpastatin mRNA abundance(p＜0.05)at 1mM Mg.Supplementation of epirenamine improved the gene expression ofμ-calpain and calpastatin(p＜0.05)comparing with the control,and the treatment with 200 ng/ml epirenamine had higher calpastatin mRNA abundance(p＜0.05) than with 50 ng/ml epirenamine,but there was not significant difference of the abundance ofμ-calpain mRNA among the treatments supplemented epirenamine.The treatment with 0.5 mM Mg+100ng/ml epirenamine had significantly higherμ-calpain mRNA abundance. The concentration of cAMP was affected significantly by supplementation of epirenamine, and the treatment with 100 ng/ml epirenamine had higher concentration of cAMP(p＜0.05) than with 50 ng/ml epirenamine.The degradation of protein was significant in treatment with 1 mM Mg or 200 ng/ml epirenamine.Experiment 6:The rat L6 myoblast and E64,which inhibits the activity ofμ-calpain, were used to investigate how the magnesium supplemented affects the tenderness.The experiment had four treatments:control,1 mM Mg,10μM E64,and 1 mM Mg+10μM E64.Detection of the abundance ofμ-calpain and calpastatin mRNA and protein concentrations ofμ-calpain and nebulin indicated that supplementation of Mg increased theμ-calpain mRNA abundance(p＜0.05)andμ-calpain(p＜0.05)and nebulin 200 kDa subfragment(p=0.096)protein concentration,whereas the protein concentration of 200 kDa nebulin was reduced significantly by supplementation of E64.In conclusion,data reported here demonstrated that:1)there was a significant difference of WBSF between Landrace and Taihu pigs,and the abundance ofμ-calpain mRNA had a negative correlation(p＜0.05)with the WBSF of LM.2)The abundance ofμ-calpain mRNA and tenderness may be improved by reducing concentration of DE and CP in diets. 3)Transportation stress affected pork quality,with TS decreasing tenderness of pork especially,and this may be related to increasing the abundance of calpastatin mRNA with TS.Supplementation of MgAsp made the serum Mg higher,improved the pork quality, and alleviated the negative effects of transportation stress on meat quality,especially for tenderness,and this may be due to that supplementation of Mg increased the abundance ofμ-calpain mRNA.4)Supplementation of Mg improved significantly the abundance ofμ-calpain mRNA and protein,and had a trend of increasing the hydrolysis product,and this suggested that it was one of mechanisms of improving the pork tenderness by Mg that supplementation Mg increased the gene expression ofμ-calpain.更多还原