Comparison Of Gastrointestinal Microbial Composition And Structure In Goat Differing In Utilization Efficiency Of Feed Nitrogen

The utilization efficiency of nitrogen in ruminants is relative low,which lead to a large amount of nitrogen is not absorbed by the body,but excreted into the environment.And,there is a serious shortage of protein feed resources in China,so it is of great significance to improve the utilization efficiency of nitrogen of ruminants.It is a complex microbial community in the rumen,and the microorganism is one of the important factors that affect the nitrogen utilization efficiency of ruminants.However,the Microbial flora in gastrointestinal tract with difirirent utilization of nitrogen in Nubian goats remains unknown.The aim of this study was to exploring the correlation between the microorganisms across gastrointestinal tract and the utilization efficiency of nitrogen.30 healthy goats were chosen as the experimental animals from a flock of goats with the same age,gender,genetic background,feeding management,and raised under the same environmental condition.Each of the 30 goats was housed in separated pens during the30-day pre-feeding and 6-day metabolism experiment.All goats were fed the same total mixed ration containing 55% forage and 45% concentration with free access to water.Every day in the period of 6-day metabolism experiment,all feces and urine were collected,the daily feed intake and residual intake were recorded within six days.At the next morning following the metabolism experiment,all goats were slaughtered at the special spot,the uncontaminated digesta samples were collected in EP tube immediately from rumen,abomasum,jejunum,cecum and colon respectively.Then,all samples were frozen in liquid nitrogen.The p H values of the rumen contents were determined on sampling spot.Accoding to the data of metabolic test,the mean and standard deviation?SD?of utilization efficiency of nitrogen was calculated.Then,the goats whoes standard deviations was above the mean of the utilization efficiency of nitrogen were chosen as the high nitrogen utilization group?HNU,Nitrogen metabolism > mean+0.5*SD?,and standard deviations was below the mean of the utilization efficiency of nitrogen were chosen as low nitrogen utilization group?LNU,Nitrogen metabolism < mean-0.5*SD?.The GIT homogenized digesta acquired from HNUand LNU group were used for DNA extraction.The DNA acquired were used as template,the microbial universal primers 515F?5'-GTGCCAGCMGCCGCGGTAA-3'?and 806R?5'-GGACTACHVGGGTWTCTAAT-3'?were used to amplify 16 S r DNA V4 hyper variable regions of the total microbial genomic.Then the products were sequenced on Illumina Hiseq2500 Sequencing Platform and the QIIME pipeline software?version 1.8.0?was used to analyse the reads acquired from sequencing.Copy numbers of seven ruminal bacteria associated with protein degradation were measured using real-time PCR,and the difference between groups was analyzed.Results:?1?The utilization efficiency of nitrogen of the thirty goats was highly variation across the individual goats?from 20.71% to 67.47%?.The difference of utilization efficiency of nitrogen between the groups was extremely significant?P < 0.000?,the average nitrogen utilization efficiency of HNU and LNU group was 56.17%±5.72% and 32.94%±7.34%?mean ± SD?.?2?At the phylum level,The most abundance phylum in rumen was Bacteroidetes?HNU61.37%,LNU 59.91%?,the secondary phylum was Firmicutes?HNU 32.11%,LNU30.75%?,and the total abundance of Bacteroidetes and Firmicutes reached to 90%.In abomasum,the majority of the sequences obtained belonged to Bacteroidetes?HNU45.59%,LNU 42.89%?,Firmicutes?HNU 29.16%,LNU 28.42%?and Proteobacteria?HNU 10.66%,LNU 17.66%?.Firmicutes?HNU 61.70%,LNU 65.57%?was the dominated phylum in jejunum,following were Lentisphaerae?HNU 7.91%,LNU 8.16%?,Bacteroidetes?the average relative abundance was 7.72%?and Tenericutes?the average relative abundance was 7.04%?.Firmicutes and Bacteroidetes were dominant bacteria phyla in colon,the average relative abundance was 63.76% and 20.99% respectively,and the relative abundance of Euryarchaeota in HNU was significant higher than that in LNU group?P=0.047?.Firmicutes and Bacteroidetes were dominant bacteria phyla in colon,the average relative abundance was 59.83% and 20.80% respectively,and the relative abundance of Cyanobacteria?P=0.042?in HNU was significant higher than that in LNU group?P=0.047?.?3?At the genus level,the relative abundance of ruminal bacterial Prevotella₁?P=0.045??Succiniclasticum?P=0.013??p-1088-a5_gut_group?P=0.044?in LNU were significant higher than that in HNU group,however the relative abundance of Ruminococcus₁?P=0.018?? Lachnospiraceae_XPB1014_group?P=0.014?? ?Ruminococcaceae_UCG-004?P=0.005?? Ruminobacter?P=0.024??Family_XIII_UCG-002?P=0.029?were significant lower than that in HNU group,within them the relative abundance of Prevotella₁ was highest?average 17.63% and 12.02% inHNU and LNU respctively?.In abomasum,the relative abundance of Eubacterium_nodatum_group?P=0.030?and Methanobrevibacter?P=0.044?in LNU were significant lower than that in HNU group,yet the relative abundance of Anaerovibrio?P=0.026?was significant higher than that in HNU group.In jejunum,the relative abundance of Ruminococcus₂?P=0.03?and Tyzzerella₃?P=0.05?in LNU was extremely higher than that in HNU.In colon,the relative abundance of Anaeroplasma?P=0.042?and Lachnospiraceae_UCG-009?P=0.014?in LNU was extremely higher than that in HNU.In cecum,the relative abundance of Prevotellaceae_UCG-004?2.52%?and Thalassospira?P=0.05?in LNU were higher than that in HNU?1.47%,P=0.032?.?4?Correlation analysis between the utilization efficiency of nitrogen and relative abundance of the bacterial composition across GIT found that the relative abundance of ruminal Ruminococcus₁?R=0.621??Ruminobacter?R=0.536?and Anaeroplasma?R=0.517??R=0.580?had a positive relationship with the utilization efficiency of nitrogen;Succiniclasticum?R=-0.55?in rumen,Anaerovibrio?R=-0.548?in abomasum,Candidatus_Saccharimonas?R=-0.75??Ruminococcus₂?R=-0.546?and Tyzzerella₃?R=-0.568?in jejunum,Lachnospiraceae_UCG-009?R=-0.75?and Blautia?R=-0.588?in colon had a negative relationship with the utilization efficiency of nitrogen.?5?The results of RT-PCR revealed that copies of Butyrivibrio_fibrisolvens?P=0.002?in HNU was extremely significant higher than that in LNU,Fibrobacter_succinogenes?P=0.022?and Ruminococcus_sp._HUN007?P=0.01?was significant higher than that in LNU,Prevotella ruminicola,Streptococcus bovis,Succinivibrio dextrinisolvens and Ruminococcus flavefaciens had no differences between two groups.In summary,this study found,there was an obvious difference in the efficiency of Nitrogen utilization among individuals;the structure of Microorganism across gastrointestinal tract with different efficiency of Nitrogen utilization were significant difference,the difference in the rumen was the most obvious and there was a certain correlation between some microbes and the efficiency of Nitrogen utilization;this experiment aslo found that Fibrobacter_succinogenes ? Butyrivibrio_fibrisolvens ?Ruminococcus_sp._HUN007 in rumen can help the utilization of nitrogen by the host.