| We are facing with challenges of lacking high quality forage,vast stalk resources wasted and continuously environmental protection pressure,so the objective of current study was to evaluate the effects of corn silage levels on changes of rumen fluid microbiota and its motabolome in Holstein heifers,under the condition of limited feeding with the same ADG,to support increasing the feed efficiency,reducing heifer cultivating cost and promoting the quality of dairy industry.24 half-sib Holstein heifers(Father ID:11106002;8-10 month of age,253±29 kg of BW at the beginning of experiments)were chosen for a four-weeks feeding trial following after 28 days’pre-experimental period.In order to reduce the potential interference from forage types and sources,the whole corn silage was used as the sole forage source.During the pre-experimental period,all the heifers were fed with adaptation diet containing 50%corn silage(DM basis).At the end of the pre-experiment,heifers were sorted in equal numbers under the randomized complete block design to one of the four treated diets with different corn silage levels(20%,40%,60%,80%of DM base,named S20,S40,S60,S80 respectively).All diets were provided as TMR with the similar intake of ME and CP.On the last day of feeding trial,rumen fluid was sampled at 3 to 4h after morning feeding.The 16S and 18S ribosome RNA gene V3-V4 region,ITS1 region of 24 rumen fluid samples were sequenced with the MiSeq platform of Illumina company.According to the rumen fluid bacterial flora sequencing result,Bacteroidetes,Firmicutes and Proteobacteria were the predominant phylums,with the sum relative abundance above 94%;Fibrobacteres and Spirochaetae,Lentisphaerae and SHA-109 were the sub-predominant phylums with each relative abundance above 0.5%.In the known genus level,Prevotella and Succiniclasticum were the predominant bacteria,with the sum relative abundance over 34%;as the sub-predominant genus,the relative abundance of Butyrivibrio,Ruminococcus,Fibrobacter,Selenomonas etc.were all above 0.5%;although other genus could be detected,the relative abundance of which were very low.The diversity(Chaol index)and richness(Shannon index)of bacteria were significantly increased with the corn silage level rising,the diversity and richness of S20 were significantly lower than that of S80(P<0.05)while there was no significantly difference between S40 and S60.As the corn silage level rising,the relative abundance of Fibrobacter,Succinimonas,Succiniclasticum,Anaeroplasms known as cellulolytic bacteria were increased linearly(q<0.05).Contrast with S20 treated group,the relative abundance of that four genus cellulolytic bacteria in S80 were increased significantly,while there were no significant different in S40 and S60.The relative abundance of other cellulolytic bacteria,such as Fibrobacter succinogenes,Butyrivibrio fibrisolvens were quite low.In total,contrast with the traditional high forage feeding model,if the forage level was not lower than 40%,the richness,diversity and the relative abundance of cellulolytic bacteria in rumen bacterial flora would be a little lower,but there were no significant difference.According to the rumen fluid protozoal flora sequencing result,Alveolata was the absolutely predominant phylum,with the relative abundance above 99.99%.In the genus level,Entodinium,Ostracodinium and Isotricha were the predominant protozoa,with the sum relative abundance above 93.78%;as the sub-predominant genus,the relative abundance of Diploplastron,Dasytricha and Pseudoentodinium were all above 0.5%;although other genus could be detected,the relative abundance of which were very low.The diversity of protozoa was increased with the corn silage level rising.and other alpha diversity indexes(such as richness)were increased but no significant different among four treated groups(P>0.05).As the corn silage level rising,the relative abundance of Entodinium known as starch stimulating protozoa were decreased linearly(q<0.05)and the relative abundance of Ostracodinium and Polyplastron were increased linearly,which plays important role in fiber degradation process.According to the rumen fluid fungal flora sequencing result,Neocallimastigomycota and Ascomycota were predominant phylum,with the sum relative abundance above 94.69%.Piromyces,Caecomyces,Neocallimastigomycota and Cyllamyces were the predominant fungi,with the sum relative abundance above 51.55%;as the sub-predominant genus,the relative abundance of Anaeromyces,Mortierella and Orpinomyces were above 0.5%,even though other genus could be detected,the relative abundance of which were very low.The alpha-diversity indexes of fungal flora changed in quadratic with the com silage level rising(S40 or S60 was the peak),but no significant different among four treated groups(P>0.1).As the corn silage level rising,the relative abundance of Neocallimastigomycota was increased linearly(q = 0.03),while that of Basidiomycota and Chytridiomycota were decreased linearly(q<0.05),but no significant different among four treated groups(P>0.05);and that of Ascomycota was decreased linearly(q<0.01)and had significant different among treated groups(P<0.01).In the genus level,the relative abundance of Neocallimastix,Kernia and Pseudeurotium were decreased linearly(q = 0.01),while Neocallimastix had no significant difference among treated groups like the other two genus(P = 0.03).The relative abundance of Cyllamyces was significantly increased linearly(q = 0.01)with the corn silage level rising.In total,the corn silage level lower than 40%would have inhibiting effect on the growth of fungi,thus S40 could offer a better fermenting condition for rumen fungal flora.According to GC-TOF/MS metabolome analysis result,558 valid peaks were captured in the rumen fluid from four groups,and 243 metabolites were identified and quantified basing on the LECO/Fiehn and KEGG metabolomics library.Using the OPLS-DA model with VIP>1.5 and q<0.05 as an option to identified significantly different metabolites in the rumen fluid by contrasting every two groups,16 different metabolites were obtained.As the corn silage level rising,six metabolites(such as 3,4-Dihydroxyphenylacetic acid)were linearly decreased(q<0.001 to q=0.032),five metabolites(such as hydrocinnamic acid)were linearly increased(q<0.001 to q = 0.019),pyruvic acid was quadratic decreased with S40 group had the highest value(q = 0.001).The correlation analysis between microbiota and its metabolome revealed that six microbe genus significantly changed with the corn silage level rising and eight different metabolites between treated groups had positive or negtive correlations.Studying the effects of dietary corn silage levels on the correlations of significant changed microbiota flora and its highly related metabolites was aiming to establish a regulatory mechanism model of dairy heifer rumen micro-ecology.As the results shown,when the dietary corn silage level not lower than 40%under the condition of limited feeding model,the richness and diversity of microbiota flora and its’ metabolic function were no significant difference with traditional feeding method. |
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