Identification Of Pig Gut Microbiota For Fiber Digestibility By 16S RRNA Gene Sequencing

China has reared a large number of pigs in the word,which were mainly fed with corn-soybean diet.The country cultivated land is limited with a large population base,the grain competition between human and animal has become more serious.Giving full play to the fiber tolerance of local pig breeds or bred swine breeds with local blood relationship and the unconventional feed rich in fiber to be selected to replace corn and soybean diet is the breakthrough to solve the grain competition between human and animal.Bacteria are essential for the breakdown of cellulose as animal alimentary enzymes are not able to digest most complex carbohydrates and plant polysaccharide.It is important to study the community structure of gut microbiota and understand the function of intestinal microbiota in contact with host.Traditional researches found that Cellulosilyticum,Clostridium and Ruminococcaceae were associated with fibers digestibility.However,most of the intestinal microbiota can not be isolated and culture.Therefore,it has great significance to use 16S rRNA gene sequencing technology to find more fiber-related microbiota.In this study,gut microbiota related to fiber digestion was identified by 16S rRNA gene sequence.Firstly,pigs in different physiological stages were selected to explore the dynamic distribution of gut microbiota and its correlation with apparent fiber digestibility.Gut microbiota related with fiber digestion was preliminary identified.Secondly,according to the variation of apparent fiber digestibility in Suhuai pig(Huai pig(Chinese indigenous pig,25%)and Large white(75%)),Pigs with extreme high and low apparent fiber digestibility were chosen to analyze the differences of gut microbiota and identify the microbiota related to fiber digestion.This lay a foundation for researching the intestinal microbial characteristics related to resistance of fiber in Chinese local pigs.Finally,based on the difference of resistance of fiber between Chinese and western pig breeds,using Erhualian and large white to explore the differences in fiber digestibility and bacterial structure with 4 kinds of fiber level diets,and to identify the microbiota related to fiber digestion.It provides an important clue for the systematic analysis of intestinal microbial characteristics related to fiber digestibility in local pigs.The main results are as follows:1.Dynamic distribution of the gut microbiota and the relationship with apparent crude fiber digestibility and growth stages in pigsApparent digestibility of fiber,data of 16S rRNA gene sequencing and their relationship in pigs at four growth stages(28 d,60 d,90 d and 150 d)were analyzed.The results showed that,apparent digestibility of CF and ADF was increased as pig aged and it was the highest in 150 d(P<0.01).Apparent digestibility of NDF in 90 d was higher than that in 60 d.A total of 19,875 operational taxonomic units(OTUs)were identified from all samples.Both bacterial abundance and diversity increased with age.A total of 22 phyla and 249 genera were identified from all fecal samples.Firmicutes and Bacteroidetes were the most dominant phyla in all samples.With increasing age,the proportion of TM7 and Tenericutes increased,whereas the proportion of Lentisphaerae and Synergistetes decreased.OTU numbers and Chao1 index were compared among the four groups.The bacteria abundance and diversity were increased as pig aged.The abundance of 36 genera varied with age,13,4 and 13 genera were associated with apparent digestibility of CF,NDF and ADF,respectively.Clostridium was reported to becorrelated with dietary fiber digestibility.Anaeroplasma,Campylobacter,Clostridium,Enterococcus,Methanobrevibacter,Nitrosospira,Propionibacterium,Pseudobutyrivibrio,Robinsoniella,Staphylococcus and Treponema participated in the fermentation process of CF;Methanobrevibacter,Parasporobacterium,Sporobacter and Treponema participated in the fermentation process of NDF;Anaeroplasma,Campylobacter,Caulobacter,Cloacibacillus,Enterococcus,Lactobacillus,Methanobrevibacter,Nitrosospira,Propionibacterium,Pseudomonas,Robinsoniella,Staphylococcus and Treponema participated in the fermentation process of ADF.2.Characteristics of Gut Microbiota in Sows and Their Relationship with Apparent Nutrient DigestibilityApparent digestibility of fiber and data of 16S rRNA gene sequencing in 9 sows were analyzed to estimate the structure and apparent nutrient digestibility of gut microbiota of sows and explore the bacteria communities related to nutrient digestibility,to compare the difference of bacterial structure and microbiota related to fiber digestibility between sows and growing pigs as well.The results showed that,the ranges of apparent digestibility values(%)of EE,CP,CF,NDF and ADF were 55.34±2.51,88.44±1.06,61.61±5.23,65.83±4.02 and 54.47±3.43,respectively.Both of the apparent digestibility of CF and ADF of sows were higher than that of two stages of growing pigs(60 d and 90 d,P<0.01).The apparent digestibility of NDF of sows was higher than that of pigs in 60 d group(P<0.01).A total ofl,235,278 sequences,25 phyla and 245 genera were identified from all fecal samples.Firmicutes and Bacteroideteswerethe most dominant phyla,approximately 73%of the total sequences.Treponema,Oscillibacter and Lactobacilluswere the most dominant generas,more than 49%of the total sequences.The microbiota of sows clustered separately from the microbiota of grower pigs at the age of 28 d,60 d,90 d and 150 d.The abundance of Anaerofustis and Robinsoniellain sow fecal samples was positively correlated with apparent CF digestibility.The abundance of Collinsella and Sutterella was positively correlated with apparent NDF digestibility.The abundance of Clostridium,Collinsella,Robinsoniella and Turicibacter was positively correlated with apparent ADF digestibility.Among these genera,Robinsoniella was found to be related to NDF and ADF apparent digestibility in growing pigs.Sows have their unique gut microbial structure compared with grower pigs and some of them participate in the digestive process of different nutrients.3.Identification of fiber digestibility related microbiota based on variation of fiber digestibility within a breedThe gut microbiota development from pigs at the growth stage to sows may be affected by the development of pig organs and the improvement of physiological functions.Therefore,further experiments should be carried out to further explore the intestinal microbial mechanism of fiber utilization in Chinese pig.Suhuai pig is a new breed with 25%tolerance to Huai pig and 75%tolerance to Large white pig,so it may have separation on fiber digestibility and gut microbiota within the breed.According to the sort of apparent NDF and ADF digestibility data in 300 Suhuai female pigs at the age of 160 d,separately screening the pigs with extreme high apparent NDF and ADF digestibility(H-NDF and H-ADF)and extreme low apparent NDF and ADF digestibility(L-NDF and L-ADF)to analyze the difference gut microbiota and further identify the microbiota related to fiber digestion.Each group included 6 pigs.The results showed that the range of apparent NDF and ADF digestibility was 44.57 to 88.34 and 29.98 to 83.09,respectively.The coefficient of variation of apparent NDF and ADF digestibility was 12.08%and 18.08%,respectively.Apparent digestibility of NDF and ADF were both significantly different between high groups and low groups(P<0.01),respectively.The results showed that a total of 975 OTUs,189 genera and 14 phyla were found in all fecal samples.A total of 188,183,188 and 185 genera were separately identified in H-NDF,L-NDF,H-ADF and L-ADF groups,while 6,1,5 and 2 genera were separately specific to H-NDF,L-NDF,H-ADF and L-ADF.The H-NDF and H-ADF microbiota clustered separately from the microbiota of the L-NDF and L-ADF along principal coordinate 1,respectively.Among the 14 phyla,the abundance of Spirochaetae,Bacteroidetes and unclassified k norank was significantly different between H-NDF and L-NDF(P<0.05)and the abundance of Spirochaetae,Verrucomicrobia,unclassified_knorank and Fibrobacterewas significantly different between H-ADF and L-ADF(P<0.05).A total of 29 and 23 genera(in total 40 different genera)were found to be potential biomarkers for distinguishing in H-NDF group and H-ADF group,respectively.Twelve of these genera were uniquely enriched in both H-NDF group and H-LDF group compared with L-NDF and L-LDF groups,respectively.Seventeen and eleven of these genera were uniquely enriched only in H-NDFgroup and H-LDF group,respectively.A number of the predicted functions were associated with carbohydrate transport and metabolism.4.Identification of fiber digestibility related microbiota based on intervarietal variation of fiber digestibilityThe previous chapter based on the variation of apparent fiber digestibility in Suhuai pig identified 40 genera related to fiber digestion.it lay a foundation for researching the intestinal microbial characteristics related to resistance of fiber in Chinese local pigs.In present study,based on the difference of resistance of fiber between Chinese and western pig breeds,Erhualian fattening barrows(Er-HL,approximately 40 kg),small Large White fattening barrows(S-LW,the same body weight as the Erhualian pigs)and large Large White fattening barrows(L-LW,the same physiological stage as the Erhualian pigs,approximately 65 kg)were selected and allotted to four fiber level treatment groups including control diet group,7%bran replaced control diet group,14%bran replaced control diet group and 21%bran replaced control diet group to analyze the differences of apparent fiber digestibility and gut bacterial structure between Erhualian and Large white,and to provide an important clue for the systematic analysis of intestinal microbial characteristics related to fiber digestibility in local pigs.The results showed that apparent digestibility of fiber(NDF,ADF,CL,ADS,TDF,IDF)of Er-HL was the highest in the 7%bran replaced basal diet group(P<0.05)and the apparent digestibility of fiber was significantly higher than S-LW and L-LW(P<0.05).Then cecum and colon mucosa samples in all breeds were used for 16S rRNA sequencing,the apparent fiber digestibility,microbiome structure and microbial functional characteristics of control diet group,7%bran replaced control diet group and 21%bran replaced control diet group.The results showed that a total of 6,708,767 sequences,1,931 OTUs,311genera and 18 phyla were found in all fecal samples.There were 34 and 9 different potential biomarkers(40 in total)in the cecum and colon in Er-HL of 7%bran replaced basal diet group,respectively.Among them,Cellulosilyticum,Ruminococcaceae_UCG₀02,Ruminococcaceae_UCG₀05,Ruminococcaceae_UCG₀09,Ruminococcaceae_UCG₀10,Ruminococcus₆ and Ruminococcaceae_NK4A214_group were known as fiber degrading bacteria.At the same time,12 biomarkers of the above 40 biomarkers were the same as those of experiment 3.The functions of the above different potential biomarkers included carbohydrate transport and metabolism.These may be the main reason of high fiber digestibility of Erhualian pig.predicted functions were associated with.As mentioned above,this paper draws conclusions as follows:1.Apparent fiber digestibility,bacterial abundance and bacterial diversity of growth pigs increased with age;The abundance of 36 genera varied with age,13,4 and 13 genera were seperately associated with apparent digestibility of CF,NDF and ADF,respectively.Different gut microbiota participated in the fermentation process of different fiber types.Compared with growth pigs,sows have their unique gut microbial structure compared with grower pigs.There were 2,2 and 4 genera were positively correlated with apparent CF,NDF and ADF digestibility in sows,respectively;Robinsoniella was the same fiber digestibility related genera both in growing pigs and sows.2.Based on variation of fiber digestibility within a breed to identify fiber digestibility related microbiota,the results showed that,29 and 23 genera were found to be potential biomarkers for distinguishing in H-NDF group and H-ADF group,respectively.Twelve of these genera were uniquely enriched in both H-NDF group and H-LDF group compared with L-NDF and L-LDF groups,respectively.Seventeen and eleven of these genera were uniquely enriched only in H-NDFgroup and H-LDF group,respectively.The main metabolic predicted functions of these genera were carbohydrate transport and metabolism.3.Based on intervarietal variation of fiber digestibility to identify fiber digestibility related microbiota,the results showed that,the level of 7%bran replaced basal diet was the optimal fiber tolerance level of Erhualian pig;a total of 40 different genera were found to be potential biomarkers which related with fiber resistance.The main metabolic predicted functions of these genera were carbohydrate transport and metabolism.4.In the present study,gut microbiota related with the fiber digestibility were identified by various methods.Among them,12 genera were mutually verified between the fourth and fifth chapters.However,this conclusion need to be further analysis and verification.