| Tibetan pig is a local pig species unique to the Qinghai-Tibet Plateau mainly living in mountains,valleys,grasslands,forests and rural fields.It has provided essential meat food for Tibetan compatriots for thousands of years.Tibetan pigs are herded and bred all the year round.They live in a variety of enviroments,have a wide range of food sources,and also face harsh natural environment.Therefore,Tibetan pigs have formed excellent characteristics such as high cold resistance,hypoxia resistance,rough feeding resistance,strong anti-stress ability and good flesh quality.The state attaches great importance to the protection and development of local excellent germplasm resources.In 2017,Tibet put forward the Tibetan Pig Industry plan,and it is imperative to breed Tibetan pigs in a large scale.The growth rate of Tibetan pigs is slower,and the nutritional requirements of Tibetan pigs are significantly different from those of lean pigs,and they can digest higher dietary fiber.Tibetan pigs cannot secrete enzymes to digest dietary fiber by themselves,and it is generally believed that the digestion of dietary fiber is mainly completed by microorganisms in the large intestine.At present,there is no data to support the crude feeding tolerance of Tibetan pigs,and it is necessary to study the mechanism of the crude feeding tolerance.In this study,the grazing Tibetan pigs fed at the grazing and breeding base in Nyingchi were used as subjects,and the house-fed Tibetan pigs and commercial pigs(DLY pig)were used as controls,comparing the structure and diversity of the bacterial community in the large intestine(caecum and colon)of three types of pigs in the original state,studying the correlation of the bacterial community and obtaining the specific bacterial community in the large intestine of three types of pigs.At the same time,10 Tibetan pigs,10 house-fed Tibetan pigs and 10 DLY pigs of the same background group were fed corn-soybean meal diet with 10% crude fiber for 15 days,the apparent digestibility of dietary nutrients,especially fiber,for 3 types of pigs was compared to obtain the roughage tolerance data of grazing Tibetan pigs,and to determine the production performance and blood physiological and biochemical indexes.In the end of the collection period,all pigs were slaughtered and their chyme(from stomach,small intestine,large intestine,and rectum)was collected.The morphology of cecum and colon,as well as the activity of fiber digestive enzyme,volatile fatty acid and p H value in cecum,colon and feces were measured,the sequence of bacterial V3-V4 region was determined by 16 S r RNA high-throughput sequencing,and the structure and composition of bacteria were compared at the level of phyla,class,order,family and genus.Pearson Correlation analysis was carried out to measure the apparent digestibility of dietary fiber,finally the key bacterial community was abtained.Single-molecule real-time sequencing(SMRT)was used to determine the structure and diversity of the fungal community in the feces.One-way Anova and Pearson Correlation analysis were performed to determine the apparent digestibility of dietary fiber.The results are as follows:1.In the natural state,the bacteria in cecum and colon of grazing Tibetan pigs had higher abundance and diversity.More specifically,the relative abundance of Firmicutes was the highest relative abundance among the three types of pigs,the relative abundance of Actinobacteria(cecum and colon)and Verrucococcus(cecum)in grazing Tibetan pigs was significantly higher than that in the other two types(P<0.05),and the relative abundance of 11 genera in the cecum of grazing Tibetan pigs was significantly higher than that of house-fed Tibetan pigs and DLY pigs(P<0.05).They are Bifidobacterium,Dehalobacterium,YRC22,Sutterella,Epulopiscium,Shuttleworthia,Mogibacterium,Allobaculum,Cupriavidus,Oxalobacter,Comamonas,respectively.The relative abundance of Bifidobacteria,Dehalobacterium,Sutterella and YRC22 in the colon of grazing Tibetan pigs was significantly higher than that of captive Tibetan pigs and DLY pigs(P<0.05).2.The body weight,body weight gain,feed intake and feed gain ratio of DLY pigs at the same age was significantly better than that of Tibetan pigs.The apparent digestibility of crude fiber,neutral detergent fiber,acid detergent fiber,cellulose and hemicellulose of grazing Tibetan pigs were significantly higher than that of captive Tibetan pigs and DLY pigs(P<0.05).The apparent digestibility of crude fat,crude protein and gross energy of DLY pigs was the highest,and that of grazing Tibetan pigs was the lowest(P<0.05).Blood physiological and biochemical indexes showed that grazing Tibetan pigs had blood cells adapted to high altitude hypoxia(P<0.05),and DLY pigs had better blood metabolism(P<0.05).3.The activities of cellulase and hemicellulase in cecum,colon and feces of grazing Tibetan pigs were significantly higher than that of the captive Tibetan pigs and DLY pigs(P<0.05).The p H values of cecum,colon and feces were neutral and slightly acidic,but there was no significant difference among the three types of pigs(P>0.05).The contents of acetate,propionate,and butyrate in cecum,colon and feces of grazing Tibetan pigs were significantly higher than those of captive Tibetan pigs(P<0.05).Cellulase was significantly positively correlated with the apparent digestibility of dietary fiber(P<0.05),cecal and fecal hemicellulase was significantly positively correlated with the apparent digestibility of dietary crude fiber and hemicellulose(P<0.05),and cecal acetate content were significantly positively correlated with the apparent digestibility of dietary hemicellulose(P<0.05).4.From the stomach to the rectum,the microorganism of the digestive tract changed significantly.The microorganisms in the stomach were obviously different from those in the small intestine due to the influence of environmental factors.A large number of microorganisms were gathered in the small intestine which were related to the digestion of carbohydrates,proteins and fats,while the microorganisms in the large intestine were mainly related to the fermentation of residual nutrients.There was a significant positive correlation between Fibrobacteres,Fibrobacter and the apparent digestibility of dietary fiber(P<0.05),and the relative abundance in cecum and colon of grazing Tibetan pigs were significantly higher than that of captive Tibetan pigs and DLY pigs(P<0.05).5.Through three-generation sequencing,15 phyla,26 classes,48 orders,87 families,190 genera and 419 species of bacteria were identified in 18 fecal samples,and 4 phyla,13 classes,23 orders,39 families,55 genera and 58 species of fungi were also identified.Among the fecal sample,bacterial taxa(Fibrobacteres,Proteobacteria,Sucinivibrio,Sphaerochaeta,Fibrobacter,Anaerovorax,Alloprevotella Rava,Fibrobacter Insomalis,Sucinivibrio Dextrinosolvens,and Papillibacter cinnamivorans),fungal taxa(Phialemonium,Podospora,Lacrymaria,Phialemonium atrogriseum,Phialemonium inflatum,and Lacrymaria subcinnamomea)were significantly positively correlated with apparent digestibility of dietary fiber(P<0.05).The relative abundance of these microbial groups in the feces of grazing Tibetan pigs was significantly higher than that of the captive pigs and DLY pigs.6.Transtranscriptome analysis showed that there were 9 differentially expressed genes(DEGs),ZFY(zinc finger protein Y-linked),AADAT(aminoadipate aminotransferase),CLDN8(claudin 8),EIF1AY(eukaryotic translation initiation factor 1A Y-linked),KDM5D(lysine demethylase 5D),LDHB(lactate dehydrogenase B),MYL2(myosin light chain 2),PLA2G2A(phospholipase A2 group IIA)and SLC22A14(solute carrier family 22 member14)may be associated with fiber utilization in the cecum and colon.Propanoate metabolism,Glycolysis/Gluconeogenesis,Glyoxylate and dicarboxylic acid metabolism,Pyruvate metabolism,Butanoate metabolism,and so on in cecum and colon were detected by KEGG enrichment of differential genes were the important metabolic pathways for dietary fiber digestible products of grazing Tibetan pigs.In summary,the main conclusions of this study are as follows:1.The Body Weight,daily gain and feed conversion efficiency of Tibetan pigs at the same age of days were lower than those of DLY pigs.Tibetan pigs raised by grazing had strong tolerance to crude feeding,which was closely related to their feeding environment,and its digestibility of dietary fiber was also stronger than that in captive Tibetan pigs and DLY pigs.Under the same feeding conditions,the difference of species(Tibetan pigs and DLY pigs)did not affect the digestibility of dietary fiber.2.There was a strong correlation between the roughage tolerance of grazing Tibetan pigs and the intestinal microecological environment.Fibrobacteres and their subspecies were the key bacteria for the degradation of dietary fiber,and intestinal cellulase and hemicellulose provide the enzyme kinetics for the degradation of dietary fiber.3.Many genes and metabolic pathways in cecum and colon provided the absorption and metabolic power for the digestible products of dietary fiber of Tibetan pigs.Propanoate metabolism,Glycolysis/Gluconeogenesis,Glyoxylate and Dicarboxylate metabolism,Pyruvate metabolism and Butanoate metabolism in cecum and colon were important metabolic pathways for dietary fiber digestible products of grazing Tibetan pigs. |
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