| The low protein-amino acid(AA)balanced diets refer to reducing of dietary protein level by 2-4 percentage units than at recommended levels of the National Research Council(NRC)with AA supplementation.The relevant studies have shown that the low protein-AA balanced diets could increase the utilization ratio of nitrogen,and reduce feed costs and nitrogen excretion without affecting the growth performance of pigs.Trillions of commensal bacteria are harbored in the pig gastrointestinal tract(GIF),and because these commensal microbiota can play a major role in the health of their host,there is increasing interest in understanding the mechanistic basis of the interaction between the diet and gut microbes.Diets with a high level of protein may disturb the intestinal microbiota,and a high protein diet could reduce the concentrations of Clostridium coccoides,Clostridium leptum,and Faecalibacterium prausnitzii in both the ceca and colons of rats.However,information on the effect of a low protein diet on the microbial composition of the gut is limited.Therefore,the aim of this study was to investigate the effects of a long-term low protein balanced with essential AA(lysine,methionine,threonine,and tryptophan)diet on the growth performance,carcass characteristics and organ weight of pigs.Meanwhile,the present study applied a combination of 454 barcoded pyrosequencing and gas chromatography-mass spectrometry(GC-MS)based metabolic profiling to explore the changes in the microbial composition and metabolomic profile induced by the dietary protein level in the hindgut of the pig.Three parts are divided into as following:1 Effects of the dietary protein level on the growth performance,carcass characteristics and organ weight of pigsThe aim of this part was to investigate the effects of a long-term low protein balanced with essential AA(lysine,methionine,threonine,and tryptophan)diet on the growth performance,carcass characteristics,blood parameters and organ weight of pigs.Thirty-six Duroc × Landrace × Large White growing barrows(70 days of age,23.57 ± 1.77 kg)were randomly allocated to normal protein diet(NP)and low protein diet(LP)groups using a randomized block design.Each group consisted of six replicates(pens)with three pigs per pen.The pigs in the NP group were offered diets which contained 18%and 16%protein according to the nutrient requirements of the NRC(1998)during the growing(days 0-50)and finishing(days 51-100)periods,respectively,while the pigs in the LP group were offered diets which contained 15%and 13%protein during their growing and finishing periods,respectively.Experimental diets designated as LP group were balanced with some AA that are not synthesized in the body(lysine,methionine,threonine,and tryptophan)to meet the requirements of growing and finishing pigs(NRC,2012).At the age of 170 days,one pig from each pen was randomly selected for slaughtering.Slaughter weight and carcass weight were measured,dressing percentage was calculated.The backfat thickness on the shoulder,thoracic waist,waist sacrum,the 6-7th rib was measured.Liver,kidney,spleen,heart,stomach,large intestine and small intestine was segregated and weighed respectively and the blood parameters were determined.The results showed that there were no significant differences in the growth performance,carcass characteristics and organ weight between the NP and LP groups.The concentrations of total protein and globulin in the blood increased with the reduction of dietary protein.The results indicated that the decrease of dietary protein level to 3%according to the nutrient requirements of the NRC(1998)supplemented with A A did not alter the growth performance,carcass characteristics and organ weight of the growing and finishing pigs.2 Effects of the dietary protein level on the microbial composition in the hindgut of the pigThe aim of this part was to investigate the effects of a long-term low protein balanced with essential AA(lysine,methionine,threonine,and tryptophan)diet on the microbial composition in the cecum and colon of the pig.Materials and methods in this experiment are based on Experiment 1.At the age of 170 days,the digesta in the hindguts of the pigs were collected for microbial analysis.Pyrosequencing of the V1-V3 region of the 16S rRNA genes showed that the dietary treatment in the condition of this study only modulated several genera without changing the diversity of gut microbiota.At the genus level,LP diet significantly decreased the relative abundance of Lactobacillus in the cecum,and Streptococcus in the colon(P<0.05);however,the relative abundance of Prevotella and Coprococcus in the LP group was significantly higher than in the NP group in the cecum,and Sarcina,Peptostreptococcaceae incertae sedis,Mogibacterium,Subdoligranulum,and Coprococcus was higher in the colon(P<0.05).No differences were found in the diversity indices and richness estimators of the cecal and colonic microbiota between the two dietary groups.The results indicated that the decrease of dietary protein level to 3%according to the nutrient requirements of the NRC(1998)supplemented with AA may modulate the microbial composition of the hindgut.3 Effects of the dietary protein level on the microbial metabolism and metabolomics profile in the hindgut of the pigThe aim of this part was to investigate the effects of a long-term low protein balancedwith essential AA(lysine,methionine,threonine,and tryptophan)diet on the microbial metabolites in the hindgut and metabolomics profile in the colon of the pig.Materials and methods in this experiment are based on Experiment 1.At the age of 170 days,the digesta in the hindguts of the pigs were collected for microbial metabolites analysis and the colonic digesta for metabolomic analysis.The results showed that the LP diet significantly increased the concentrations of microbial crude protein(MCP)and crude mucin in the colon(P<0.05),and decreased methylamine in the colon and the concentrations of isobutyrate,isovalerate,branched-chain fatty acids(BCFAs)/short-chain fatty acids(SCFAs)in the cecum(P<0.05)compared with NP diet.However,there were no significant differences in the concentrations of ammonia-nitrogen,tryptamine,putrescine,cadaverine,or tyramine between the NP and LP groups.The GC-MS analysis showed that nine of the compounds(xanthine,hypoxanthine,stearic acid,glyceric acid,methyl phosphate,malic acid,leucine,glutamic acid,and tyrosine)were enriched,while benzoate and lyxose were reduced in the LP group,when compared with the NP group.Further metabolic pathway enrichment analysis showed that the different protein levels of the diet mainly affected the phenylalanine metabolism;glycine,serine,and threonine metabolism;the citrate cycle;pyruvate metabolism;and the alanine,aspartate,and glutamate metabolism.Moreover,the correlation analysis of the combined datasets revealed some potential relationships between the colonic metabolites and certain microbial species.These results suggest that a low protein diet may modulate the metabolites of the hindgut,however,potential roles of this modulation to the health of pigs remains unknown. |
