Mechanism Of Fat Deposition Regulated By Intestinal Microbiota In Growing-finishing Pigs

The gastrointestinal tract of mature pigs is colonized with trillions of diverse microbial communities that closely related to the metabolism of nutrients,immune response,and growth and development in pigs.Amounts of studies have indicated that the intestinal microbiota plays a pivotal role in the digestion,absorption,and metabolism of lipids,affects the metabolism of lipid in different tissues,and regulates the fat deposition in liver,subcutaneous adipose tissue,and muscle.In the pig industry,the content of subcutaneous fat(SAT)and intramuscular fat(IMF)is important factor affecting pork quality,while pork quality is one of the most important economic indicators that directly affect consumers’sensory characteristics and quality grades of meat products.The Laiwu pig is one of the excellent native Chinese breeds characterized by high IMF content and good meat quality,and its intestinal microbiota are important sources for improving pork quality of commercial pigs.Here,we compared the difference of gut microbiota composition as well as their metabolic functions between Laiwu pigs and commercial Duroc×(Landrace×Yorkshire)(DLY)pigs by 16S r DNA sequencing.Using Fecal microbiota transplantation(FMT),the fecal microbiota of healthy Laiwu pigs were transplanted into DLY pigs to investigate the effects of intestinal microbiota on pork quality and fat deposition in growing-finishing pigs.Integrated multi-omics analysis was performed to screen the core candidate strains which involved in the regulation of fat deposition.The core candidate strains were transplanted to SPF C57BL/6j mice to analyze the mechanisms of their regulation on fat deposition.The main studies and findings are:1.Comparative study on the differences in gut microbial composition and their function between Laiwu pigs and Duroc×(Landrace×Yorkshire)pigsThe feces were collected from healthy Laiwu pigs and DLY pigs and analyzed by16S r DNA sequencing.Comparative analysis of microbiome was performed to investigate the difference of microbial composition in Laiwu pigs and DLY pigs.The functional different of microbiome was analyzed by PICRUSt.The result showed a distinct difference in microbial composition between Laiwu pigs and DLY pigs.The diversity and richness of gut microbiome in Laiwu pigs were significantly higher than DLY pigs.The most dominant bacterium was Bacteroides in Laiwu pigs and shown a significantly higher proportion(55.4%versus 29%,P<0.01)compared to DLY pigs.The relative abundance of Firmicutes in Laiwu pigs was significantly lower than in DLY pigs(38.0%versus 60.4%,P<0.05).Microbiota related to the biosynthesis of secondary metabolites,amino acid metabolism,energy metabolism and carbohydrate metabolism were significantly enriched in Laiwu pig.These results suggested that the distinct differences of microbial composition and their function between Laiwu pigs and DLY pigs might be important factors on IMF content.2.Impacts of microbiota on pork quality and lipid metabolism were studied by fecal microbiota transplantationTo investigate the relationship between intestinal microbiota and fat deposition,fecal microbiota from healthy Laiwu pigs was transplanted into DLY pigs.A total of 32 DLY piglets with similar weight(29.6±0.18 kg)were randomly assigned to 4 treatment groups,received sterile saline(control group,Ctrl),sterile fecal suspension(sterilized fecal microbiota transplantation,SFMT),10~7 CFU/m L fecal suspension(low-dose of fecal microbiota transplantation,LFMT),and 10~8 CFU/m L fecal suspension(high-dose of fecal microbiota transplantation,HFMT)by oral gavage once every other day until reached market weight.The results indicated that the HFMT selectively enhances fat deposition in longissimus dorsi but decrease in backfat thickness compared with control group(P<0.05).Transmission electron microscopy(TEM)analysis and Oil red-O staining of liver indicated that the hepatic tissue of HFMT displayed a significant increase in fat deposition.The H&E staining of small intestine showed that HFMT significantly increased villi heights and the ratios of villi heights to crypts depths in the duodenum,jejunum,and ileum(P<0.05).We determined the concentration of fecal short chain fatty acids(SCFAs)using gas chromatography analysis.It is noteworthy that the concentration of isobutyric acid and isovaleric acid was significantly higher in HFMT pigs than that in control pigs(P<0.05).These results suggested that microbiota can regulate IMF and subcutaneous fat deposition in pigs,which may relate to the production of SCFAs by microbial fermentation.Integrated analysis of microbiome,lipidome and proteome to screen the core candidate strains regulating fat deposition in pigs.The results of microbiome showed a bloom of Bacteroides and reduction in Firmicutes in HFMT,which were closer to the property of microbial composition in Laiwu pigs,suggesting that FMT caused shifts from DLY pigs to Laiwu pigs in the bacterial community structure.To determine whether the increase of fat deposition is correlated with shifts of lipid metabolites,we integrated lipid metabolomic data from the small intestine,plasma,liver,and muscle to investigate the effects of HFMT on lipid metabolism in different tissues.The results of multi-tissues lipidomic analysis revealed that HFMT could significantly increase the expression level of fatty acyls and glycerophospholipids in liver and muscle,decrease the expression level of glycerolipids.Moreover,proteomic analysis of small intestine and muscle showed that HFMT increased the expression levels of protein-associated with lipogenesis(FABP4、FASN、FADS1)and decreased the expression level of protein-related to lipid metabolism(ACADM).Furthermore,combining correlation analysis,we screened four bacterial species(B.uniformis,S.globosa,H.saccharovorans,and P.piscolens)which may involve in the regulation of fat deposition.3.Transplantation of core candidate strains into SPF C57BL/6j mice to analyze the mechanisms of their regulation on fat depositionWe inoculated SPF C57BL/6j mice with the 4 core candidate strains respectively or their mixture(Mixed)to validate the effects and mechanisms of bacteria on fat deposition.The results showed that transplantation of mixed core candidate strains can improve the IMF,body fat,and SAT content of mice.P.piscolens transplantation increased SAT,VAT,and body fat content,but no significantly improve IMF of mice.This result suggests that the regulation of bacteria on fat deposition probably shows tissue-specific.In the liver of the mice transplanted with mixed core candidate strains,the expressions of genes-associated with lipid metabolism(Ppara,Cpt1a,Cpt1b,and Pgc1a)and lipogenesis(Pparg and Dgat2)were significant increased(P<0.05),suggesting that core candidate strains promote lipid turnover in the liver of mice.In the muscle of the mice transplanted with mixed core candidate strains,the expressions of genes-associated with lipogenesis(Cd36,Fasn,Acaca and Dgat2)were increased(P<0.05),while no significant difference were observed in the expressions of genes-related to lipid metabolism.These results indicated that transplantation with mixed core candidate strains can increase fat deposition in muscle by improving the expressions of genes-associated with lipogenesis,including Cd36,Fasn,Acaca and Dgat2.In conclusion,the gut microbiota is involved in regulating of IMF deposition in growing-finishing pigs,and its mechanism is to promote IMF deposition by up-regulating the expression of lipogenesis-associated genes Cd36,Fasn,Acaca and Dgat2 in muscle.This study provides an important theoretical basis for improving pork quality by developing the new functional microbiota.