The Relationship Between μ-Calpain, Calpastain And Pork Tenderness And The Study On Nutritional Regulation Of Its Expression

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.