Effects Of Creatine Monohydrate And Guanidinoacetic Acid Supplementation On Meat Quality Of Finishing Pigs And Mechanisms Involved

The meat quality characteristics are related to the physiological state of muscle post mortem.It is confirmed that creatine monohydrate?CMH?supplementation in finishing pigs could increase the phosphocreatine content in the muscle,and delay the glycolysis post mortem to improve the pork quality.Guanidinoacetic acid?GAA?is the only immediate precursor for creatine,which could be conversed to creatine in body.The objective of this study was to evaluate the effects of CMH and GAA supplementation on growth performance,slaughter performance,meat quality,creatine synthesis and transport and postmortem energy metabolism of finishing pigs.In addition,research on differential protein profile in longissimus dorsi muscle and differential metabolites profile in plasma were mainly carried out by proteomics and metabolomics technology,to preliminarily explore the possible mechanism.1 This part of study was to investigate the effects of CMH supplementation on slaughter performance,meat quality and postmortem energy metabolism of finishing pigs.In total,48healthy Duroc-Landrace-Large White cross castrated pigs??90 kg?were randomly allocated into two treatments,and fed either a CHM-free basal diet or a basal diet with CMH supplementation?0.8%?for 14 days.At the end of the experiment,six pigs from each treatment were weighed individually and slaughtered.The muscle from longissimus dorsi was collected to evaluate the meat quality and energy metabolism-related parameters.The results were shown as follow.CMH supplementation did not affect the loin eye area,back fat depth and dressing percentage?P>0.05?.Dietary CMH induced an increase of pH_45min and lower drip loss and coking loss?P<0.05?,but had no effect on L*value,a*value,b*value and share force?P>0.05?.There were no difference on moisture,crude fat,crude protein and ash concentrations between two groups?P>0.05?.In addition,CMH supplementation did not affect cohesiveness and springiness of longissimus dorsi?P>0.05?,but had a tendency to increase the hardness,gumminess and chewiness?P>0.05?.In the longissimus dorsi at 45 min post mortem,dietary CMH increased the creatine,ATP,ADP springiness,chewiness in longissimus dorsi,and cohesiveness and gumminess in semitendinosus?P>0.05?.Dietary CMH and GAA had no effect on T22 peak area of longissimus dorsi and semitendinosus?P>0.05?.?2?CMH supplementation increased the creatine and phosphocreatine concentrations in longissimus dorsi?P<0.05?,and increased the level of phosphocreatine in semitendinosus?P<0.05?,and had no effect on creatine and phosphocreatine concentrations in liver and kidney?P>0.05?,and increased the activity of CK in longissimus dorsi?P<0.05?,but not in semitendinosus?P>0.05?.Dietary GAA increased the creatine and phosphocreatine concentrations in longissimus dorsi?P<0.05?,phosphocreatine concentration in sermitendinosus?P<0.05?,creatine concentration in liver?P<0.05?,but had no effect on creatine and phosphocreatine concentrations in kidney?P>0.05?,and had a tendency to increased the activity of CK in longissimus dorsi?P>0.05?,but not in semitendinosus?P>0.05?.Dietary CMH and GAA decreased the expression of arginine:glycine amidinotransferase?AGAT?in kidney?P<0.05?,and no differences were observed between groups for the expression of AGAT in longissimus dorsi,semitendinosus,and liver?P>0.05?.CMH supplementation increased the expression of S-adenosyl methionone:guanidinoacetate methyltransferase?GAMT?in longissimus dorsi,semitendinosus,and kidney?P<0.05?,and GAA supplementation increased the expression of GAMT in liver?P<0.05?,and had a tendency to increase the expression of GAMT in longissimus dorsi and kidney?P>0.05?.CMH supplementation increased the expression of creatine transporter?CreaT?in longissimus dorsi,liver and kidney?P<0.05?,and GAA supplementation increased the expression of CreaT in longissimus dorsi?P<0.05?,and had a tendency to increase the expression of CreaT in other tissues?P>0.05?.?3?CMH and GAA supplementation increased the level of ATP in longissimus dorsi and semitendinosus?P<0.05?,but had no effect on ADP and AMP concentrations?P>0.05?.Dietary CMH had a tendency to decrease the lactic acid content and to decrease the activity of HK?P<0.05?,and decreased the activity of LDH significantly?P>0.05?,but had no effect on the level of glycogen and GP,and the activity of PK?P>0.05?in longissimus dorsi.Dietary CMH increased the glycogen concentration?P<0.05?,and had a tendency to decrease the lactic acid content?P>0.05?,but had no effect on the level of GP,the activity of LDH,HK and PK?P>0.05?in semitendinosus.GAA supplementation decreased the lactic acid content and the activity of HK?P<0.05?,and had a tendency to decrease the activity of LDH?P>0.05?,but had no effect on the level glycogen and GP,and the activity of PK?P>0.05?in longissimus dorsi.GAA supplementation increased the glycogen concentration?P<0.05?,and decreased the level of lactic acid?P<0.05?,but had no effect on the level GP,the activity of LDH,HP and PK?P>0.05?in semitendinosus.Dietary CMH increased the expression of adenosine 5'-monophosphate-activated protein kinase?2?AMPKa2?in longissimus dorsi and glycogen synthase?GYS?in semitendinosus?P<0.05?,and had a tendency to increase the expression of glucose transporter type 4?GLUT4?and GYS in longissimus dorsi and AMPKa2 and GLUT4 in semitendinosus,.Dietary GAA increased the expression of AMPKa2 in longissimus dorsi?P<0.05?,and had a tendency to increase the expression of GLUT4 and GYS in longissimus dorsi and AMPKa2,GLUT4and GYS in semitendinosus?P>0.05?.3 This part of study was to investigate the effects of CMH and GAA supplementation on difference of protein profiles of muscle in finishing pigs.The experiment design was the same with Chapter 2.Proteins from longissimus dorsi were isolated by differential centrifugation and were separated by two-dimensional gel electrophoresis?2-DE?.Then the proteins were further identified by mass spectrum?MS?analysis.Compared with control group,CMH supplementation up-regulated 17 different proteins,and GAA supplementation up-regulated 11 different proteins and down-regulated 7 different proteins.After Gene Ontology?GO?analysis,the differential proteins in CMH group were related to several biological processes as follow:muscle filament,muscle contraction,response to hydrogen peroxide,regulation of ATPase activity,sarcomere organization,cellular component movement,hydrogen peroxide catabolic process,glycolysis and so on.The differential proteins in CMH group were related to several cell components as follow:cytosol,extracellular vesicular exosome,myofibril,cytoskeleton,muscle thin filament tropomyosin,sarcomere,Z disc,mitochondrial matrix,myosin filament and so on.The differential proteins in CMH group were related to several molecular functions as follow:identical protein binding,actin binding,microfilament motor activity,tropomyosin binding,peroxidase activity,structural constituent of muscle,alkyl hydroperoxide reductase activity,protein kinase C binding and so on.After Kyoto Encyclopedia of Genes and Genomes?KEGG?pathway analysis,the differential proteins in CMH group were related to several pathways as follow:glycolysis/gluconeogenesis,tight junction,HIF-1 signaling pathway and thyroid hormone signaling pathway.After GO analysis,the differential proteins in GAA group were related to several biological processes as follow:muscle filament sliding,cellular component movement,glycolysis,regulation of muscle contraction,sarcomere organization,purine ribonucleotide catabolic process,regulation of interleukin-1-mediated signaling pathway,gluconeogenesis and so on.The differential proteins in GAA group were related to several cell components as follow:cytoskeleton,extracellular vesicular exosome,muscle thin filament tropomyosin,cytosol,filamentous actin,extracellular space,pyruvate dehydrogenase complex,Z disc and so on.The differential proteins in GAA group were related to several molecular functions as follow:structural constituent of muscle,protein binding,protein kinase C binding,3-hydroxyisobutyrate dehydrogenase activity,cytoskeletal protein binding,FATZ binding,troponin C binding,pyruvate dehydrogenase?acetyl-transferring?activity,protein kinase C inhibitor activity and so on.After KEGG pathway analysis,the differential proteins in GAA group were related to several pathways as follow:glycolysis/gluconeogenesis,HIF-1 signaling pathway,purine metabolism,pentose phosphate pathway and Citrate cycle?TCA cycle?.4.This experiment was designed to investigate the effects of CMH and GAA supplementation on metabolism of finishing pigs by using metabolomics method.The experiment design was the same with Chapter 2.The metabolites in plasma were separated and identified by gas chromatography-mass spectroscopy?GC-MS?.The results were shown as follow.CMH supplementation changed the concentrations of 21 kinds of metabolites,in which 17 different metabolites were decreased compared to control group,including branched chain amino acids?BCAA?,glucogenic amino acids,fatty acids,lactic acid and taurine,and 4 different metabolites were increased compared to control group,including glucose,lactose,glycine and creatinine.All of these metabolites were related to protein biosynthesis,galactose metabolism,BCAA degradation,ammonia recycling,taurine and hypotaurine metabolism,lipid metabolism,glutathione metabolism and bile acid biosynthesis.GAA supplementation changed the concentrations of 24 kinds of metabolites,in which 19 different metabolites were decreased compared to control group,including BCAA,glucogenic amino acids,fatty acids,lactic acid and taurine,and 5 different metabolites were increased compared to control group,including glucose,D-turanose,succinic acid,glycine and creatinine.All of these metabolites were related to protein biosynthesis,ammonia recycling,urea cycling,lipid metabolism,BCAA degradation,glutathione metabolism,arginine and proline metabolism,taurine and hypotaurine metabolism.As stated above,the conclusions are as follow:?1?CMH and GAA supplementation could improve the growth performance and meat quality of finishing pigs,and promote the absorption and transport of creatine in muscle,inhibit the biosynthesis of GAA and creatine in body,and increase the concentrations of creatine and phosphocreatine in cells.CMH and GAA supplementation could increase the ATP concentrations in muscle,and delay the glycolysis by reducing the activities of glycolytic enzyme,resulting a decreased lactic acid accumulation,and regulate the energy metabolism by activating the AMPK pathway.?2?This study investigated the effects of CMH and GAA supplementation on protein profiles of muscle in finishing pigs.In total,27 different proteins were separated and identified successfully,in which CMH supplementation affected 17 different proteins,and GAA supplementation affected 18 different proteins.After GO and KEGG analysis,CMH and GAA had the similar mechanism to affect the meat quality by regulate the expression of structural protein of muscle fibers,the energy metabolism of muscle,and the antioxidant status.But there were some differences between these two additives,in which CMH could regulate the metabolism of skeletal muscle by regulate the tight junction of muscle fibers and thyroid hormone signaling pathway,and GAA could regulate the metabolism of skeletal muscle by regulate purine metabolism and pentose phosphate pathway.?3?This study investigated the effects of CMH and GAA supplementation on metabolites profiles of plasma in finishing pigs.CMH supplementation affected 21 different metabolites in plasma,in which they were related to protein biosynthesis,galactose metabolism,BCAA degradation,ammonia recycling,taurine and hypotaurine metabolism,lipid metabolism,glutathione metabolism and bile acid biosynthesis.GAA supplementation affected 24different metabolites in plasma,in which they were related to protein biosynthesis,ammonia recycling,urea cycling,lipid metabolism,BCAA degradation,glutathione metabolism,arginine and proline metabolism,taurine and hypotaurine metabolism.