Regulation Of Porcine Skeletal Musclephenotypes And Metabolism By Gut Microbiota And Its Interaction With Nutrition

Skeletal muscle-phenotypes,intramuscular fat(IMF)content and myofiber characteristics,are the main factors determining pork eating quality.Recently,the key role of gut microbiota in regulating host physiology has been deciphered,but it remains unknown whether the gut microbiota mediates the breed-induced differed skeletal muscle-phenotypes.In addition,it is not clear whether the interaction between nutrition and gut microbiota affects skeletal muscle-phenotypes.Four trials were conducted to investigate the effects of pig breed,porcine fecal microbiota transplantation or post-weaning antibiotic exposure on gut microbiota composition,skeletal muscle-phenotypes and metabolism,as well as study the responses of pigs which harbor distinct gut microbiota to dietary starch type.The data got in our research will provide new insights into the interactions among nutrition,gut microbiota and host.Exp.1 The differences in gut microbiota composition,skeletal muscle-phenotypes and metabolism between pig breedsThe present study was conducted to compare the differences in gut microbiota composition,skeletal muscle-phenotypes and metabolism between obese Rongchang pigs(RP)and lean Yorkshire pigs(YP).5 RP and 5 YP aged 12 weeks were fed with the same diet without antibiotic,the trial lasted for 56 days.The results are as follows:1)RP had higher fat percentage and lower lean percentage as compared to YP(P<0.05),and there is a trend toward higher myofiber diameter and cross-sectional area in gastrocnemius muscle of RP than that of YP(P=0.081).The IMF content,triglyceride concentration and lipoprotein lipase(LPL)activity in gastrocnemius muscle of RP were higher than that of YP(P<0.05).2)The mRNA abundances of MYH7,ACACA,FASN,SREBF1 and LPL were higher in gastrocnemius muscle of RP than that of YP(P<0.05),CD36 mRNA abundance tended to increase in gastrocnemius muscle of RP(P=0.081),but the expression of CPT1 B and MYH4 were down-regulated in gastrocnemius muscle of RP as compared to YP(P<0.05).3)Compared with YP,RP exhibited higher Firmicutes and Spirochaetes abundances,and lower Bacteroidetes abundance(P<0.05).The plot of beta diversity index showed significant separate clustering of samples according to pig breed.4)At the phylum level,the bacterial abundance of Bacteroidetes was negatively correlated with IMF content,triglyceride concentration and MYH7 mRNA level and was positively correlated with myofiber diameter and cross-sectional area in gastrocnemius muscle(P<0.05).The bacterial abundance of Firmicutes was negatively correlated with myofiber diameter and cross-sectional area and was positively correlated with triglyceride concentration in gastrocnemius muscle(P<0.05).The bacterial abundance of Spirochaetes was positively correlated with IMF content,triglyceride concentration and MYH7 mRNA level and was negatively correlated with myofiber diameter,myofiber cross-sectional area and MYH4 mRNA level in gastrocnemius muscle(P<0.05).The bacterial abundance of Verrucomicrobia was positively correlated with triglyceride concentration in gastrocnemius muscle(P<0.05).5)At the genus level,the bacterial abundances of Ruminococcus,YRC22 and Treponema were positively correlated with IMF content,triglyceride concentration and MYH7 mRNA level,and were negatively correlated with myofiber diameter,myofiber crosssectional area and MYH4 mRNA level in gastrocnemius muscle(P<0.05).The bacterial abundances of Anaerovibrio,Succinivibrio and Prevotella were positively correlated with myofiber diameter,myofiber cross-sectional area and MYH4 mRNA level,and were negatively correlated with triglyceride concentration and MYH7 mRNA level in gastrocnemius muscle(P<0.05).The bacterial abundance of Megasphaera was negatively correlated with triglyceride concentration and MYH7 mRNA level in gastrocnemius muscle(P<0.05).The results suggested the significant differences in carcass composition,skeletal musclephenotypes and gut microbiota composition existed between obese and lean pig breed,and the bacterial abundances of predomidant phyla and genera were significantly correlated with skeletal muscle-phenotypes.Exp.2 Colonization of germ-free mice with porcine gut microbiota influences skeletal muscle-phenotypes and metabolismThe present study was conducted to investigate the effects of fecal microbiota transplantation on gut microbiota composition,skeletal muscle-phenotypes and metabolism of germ-free mice.A total of 20 germ-free newborn mice were randomly assigned to two treatments(n=10),RM and YM,which were transplanted with either Rongchang pigs derived-fecal microbiota or Yorkshire pigs derived-fecal microbiota.The trial lasted for 35 days.The results are as follows:1)Compared with YM,RM exhibited higher abundances of Firmicutes,Fusobacteria and Actinobacteria,and lower abundances of Bacteroidetes and Proteobacteria(P<0.05).The plot of beta diversity index indicated that the mice recipients shared high similarity in fecal microbiota composition with the corresponding donors,transplanting fecal microbiota into germ free mice replicated the gut microbiota composition of pig donors.2)RM had higher body fat percentage than YM(P<0.05).The cross-sectional area tended to decrease(P=0.0832),and triglyceride concentrations and LPL activity were significantly increased in gastrocnemius muscle of RM when compared to YP(P<0.05).3)There was a trend towards higher mRNA abundances of MYH7,LPL,CD36 and SREBF1 in gastrocnemius muscle of RP than that of YP(P=0.0576,P =0.0864,P=0.0812,P=0.066).The expression of ACACA was up-regulated,and the expression of MYH4 and CPT1 B were down-regulated in gastrocnemius muscle of RP as compared to YP(P<0.05).The results indicated that the porcine microbiota-associated mice model was successfully established by fecal microbiota transplantation,as the similarity in microbial community was extreme high between mice recipients and pig donors.Transplantation of fecal microbiota from Rongchang pig increased fat deposition and shifted the myofiber characteristics in skeletal muscle,indicating that gut microbiota can transfer host phenotypes across species.Exp.3 Effects of antibiotic-driven microbiota perturbation on skeletal musclephenotypes and metabolism of pigletsThe present study was designed to investigate whether muscle fat deposition and fiber characteristics in piglets are influenced via manipulating microbiota with antibiotic.A total of 18 piglets(Topics hybrid × Piétrain),aged 28 days,were fed with either a control diet or an antibiotic-containing diet(n=9;tylosin phosphate,100 mg/kg diet).Treatments were balanced for weight,gender and litter.The trial lasted for 39 days.The results are as follows:1)The two groups shared a high similarity in baseline fecal microbiota composition.After 39 days' tylosin exposure,antibiotic-treated piglets exhibited higher abundance of Firmicutes and increased Firmicutes/Bacteroidetes ratio in caecal digesta when compared to control piglets(P<0.05).The abundance of Firmicutes tended to increase in colonic digesta of antibiotic-treated piglets(P=0.069).There was a trend toward higher Bacteroidetes abundance in both caecal(P=0.056)and colonic digeseta(P=0.076)of antibiotic-treated piglets.The plot of beta diversity index showed that antibiotic administration shifted the microbiota composition in caecum and colon of piglets.2)Antibiotic administration significantly increased final weight and growth rate and decreased feed to gain ratio of weaning piglets(P<0.05).Antibiotic administration significantly increased total volume,lean volume and fat volume of carcass(P<0.05)without affecting the lean and fat percentage of carcass in weaning piglets(P>0.05).3)Antibiotic administration tended to increase serum triglyceride concentration(P=0.08),and significantly increased the IMF content in longissimus muscle of weaning piglets(P<0.05).4)Antibiotic-treated piglets had lower myofiber density,higher mRNA levels of MYH7 and MYH4 and lower mRNA level of MYH1 in longissimus muscle as compared to control pigs(P<0.05).5)Antibiotic administration significantly increased mRNA levels of ACACA,LPL and CD36(P<0.05),tended to increase gene expression of FASN(P=0.05),and tended to decrease mRNA abundance of PNPLA2 in longissimus muscle(P=0.07).6)Antibiotic administration significantly decreased the mRNA abundance of ANGPTL4 and PPARG in colonic mucosa of piglets(P<0.05).The results presented above indicated that post-weaning antibiotic exposure improved the growth performance,shifted the carcass composition,and increased muscular fat deposition and changed myofiber type distribution of piglets.The underlying mechanism of these effects is possibly related to the regulation of PPARG-ANGPTL4 in intestinal mucosa.Exp.4 Effects of baseline gut microbiota composition and dietary starch type on skeletal muscle-phenotypes and metabolism of finishing pigsA 2×2 factorial arrangement study was designed to evaluate the effects of Prevotella/Bacteroides ratio(high P/B or low P/B ratio)and dietary starch type(amylose or amylopectin)on gut microbiota,skeletal muscle-phenotypes and metabolism.12 finishing pigs with high P/B ratio(HPB)and 12 finishing pigs with low P/B ratio(LPB)were fed either control diet(C)or high amylose diet(RS)(30% high amylose maize substituted for 30% high amylopectin maize in control diet).Four treatments were formed(P/B ratio plus diet): HPB + C,LPB + C,HPB + RS,LPB + RS(n=6).The trial lasted for 38 days.The results are as follows:1)The significant differences in baseline fecal microbiota between HPB pigs and LPB pigs were observed(P<0.05).The P/B ratio was higher in HPB pigs than LPB pigs(P<0.05).2)Compared with control diet,high amylose diet significantly decreased apparent digestibility of dry matter,energy and protein,reduced ileal mucosal amylase activity and ANGPTL4 concentration(P<0.05),increased serum insulin,acetic acid and total SCFAs concentrations,up-regulated PPARGC1 A mRNA abundance in longissimus muscle and LPL mRNA abundance in semitendinous muscle,and elevated acetic acid and total SCFAs concentrations in colon digesta(P<0.05).The plot of beta diversity index showed high amylose diet significantly shifted the gut microbiota of finishing pigs.3)Compared with LPB pigs,HPB pigs exhibited lower apparent digestibility of dry matter,energy and protein,decreased serum insulin level,down-regulated mRNA abundances of ACACA,CD36,PPARGC1 A and MYH7 in longissimus muscle and lower mRNA abundances of ACACA,LPL,CD36 and MYH7 as well as decreased IMF content in semitendinous muscle,reduced ileal mucosal LPS content and colonic mucosal SLC5A8 mRNA abundance(P<0.05),increased serum ANGPTL4 and propionic acid concentrations,higher mRNA abundances of ANGPTL4,CPT1 B and PRKAA1 in longissimus muscle,and elevated ileal mucosal ANGPTL4 content(P<0.05).Compared with LPB pigs,HPB pigs exhibited higher Bacteroidetes abundance and P/B ratio in colon digesta(P<0.05).The plot of beta diversity index showed significant separate clustering of samples according to P/B ratio(P<0.05).X4)The interaction between Prevotella/Bacteroides ratio and dietary starch type significantly affected serum ANGPTL4 concentration,regulated IMF content,triglyceride level,myofiber diameter and CD36 mRNA abundance of semitendinous muscle,influenced ileal mucosal LPS content,modulated colonic mucosal PPARG,ANGPTL4 and SLC5A8 mRNA abundances,and modulated acetic acid and total SCFAs concentrations in colon digesta(P<0.05).Compared with control diet-fed LPB pigs,high amylose diet-fed LPB pigs had enhanced IMF content,triglyceride level and CD36 mRNA abundance of semitendinous muscle,elevated ileal mucosal LPS content and colonic mucosal SLC5A8 mRNA abundance,increased acetic acid and total SCFAs concentrations in colon digesta,lower serum ANGPTL4 content,reduced myofiber diameter of semitendinous muscle and decreased mRNA abundances of PPARG and ANGPTL4 in colon mucosa.However,no difference in above-mentioned parameters were observed between control diet-fed HPB pigs and high amylose diet-fed HPB pigs.In conclusion,high amylose diet shifted the gut microbiota of pigs and increased SCFAs contents in hindgut.HPB pigs exhibted lower IMF content in skeletal muscle,which might be attributed to down-regulated expression of genes related to de novo synthesis and uptake of fatty acid.Gut microbiota P/B ratio interacted with dietary stach type to significantly influence skeletal muscle-phenotypes and metabolism,high amylose diet increased intramuscular fat content,decreased myofiber size,and shifted myofiber type distribution in skeletal muscle of pigs with low P/B ratio,which might be attributed to the regulation of PPARG-ANGPTL4 in intestinal mucosa and the accumulation of acetic acid in hindgut.Collectively,the significant differences in gut microbiota composition,skeletal musclephenotypes and metabolism existed between Chinese indigenous pig breed and foreign pig breed,and these differences can be transferred from pig donors to mice recipients by fecal microbiota transplantation.Post-weaning tylosin administration shifted the gut microbiota composition,increased Firmicutes/Bacteroidetes ratio,and further influenced skeletal muscle-phenotypes and metabolism of piglets.Both gut microbiota P/B ratio and dietary starch type significantly influenced skeletal muscle-phenotypes and metabolism,and the gut microbiota P/B ratio might play a key role in determining the host responses to dietary starch type.Therefore,skeletal muscle-phenotypes and metabolism are not only related to pig breed,but also closely correlated with gut microbiota,the intramuscular fat content and myofiber characteristics in skeletal muscle of pigs can be modulated via manipulating gut microbiota composition.