Effects Of Dietary Intervention On Rumen Fermentation And Microbiota In Sheep Fed Low Quality Forage

Cereal straw is one of the most abundant biomass resources in the world.The microorganisms inhabiting the rumen of the ruminants may digest the fibrous materials of the cereal straw and the end-products are the energy source for the host.Feeding animals with fibrous materials usually stimulates the growth of lignocellulose-utilizing bacteria and improves fibrolytic potential in the rumen.Cereal straw consists of high contents of fibrous material,which may exert great effects on the fibrolytic bacteria and fiber digestion in the rumen.However,information is limited on these aspects.Therefore,the objective of this study was to seek the possibility of regulating rumen microbial community to enhance fibrolytic potential through low quality forage as dietary intervention.The study was conducted in two parts.In Part 1,we investigated in situ degradation of alfalfa hay(AH)and corn stover(CS)and temporal dynamics of microbial colonization on forage particles in the different rumen environment when animals were fed AH or CS.In Part 2,low quality forage was fed as dietary intervention to investigate nutrients digestibility,rumen fermentation characteristics,and variation of rumen microbial community during forage transition from AH to CS and vice versa.1.Forage degradation and microbial colonization under different rumen environment caused by feeding different forage sources.Three rumen cannulated Hu sheep were assigned to dietary treatment of AH and CS for 4 weeks each period,respectively,in order to investigate forage degradation and microbial colonization under different rumen environment caused by feeding AH or CS.In the last two days of each period,the samples of AH and CS in nylon bags were incubated in the rumen for 0.5,2,4,8,16,24 and 48 h to measure the rumen degradation and microbial colonization.Rumen contents were also sampled simultaneously to determine rumen fermentation variables.1.1 Effects on rumen fermentation and in situ forage degradationUnder different feeding regime,there existed significant difference in the rumen fermentation in Hu sheep.The rumen pH of sheep fed AH significantly decreased during the first half an hour after morning feeding(P<0.05),and reached its nadir at 8 h,significantly lower than CS-fed animals(P<0.05),while the pH of the CS-fed sheep was much flattered.Concentrations of total and individual volatile fatty acids(VFA)including acetic,propionic,butyric,valeric,iso-butyric,iso-valeric acids as well as acetate to propionate ratio were significantly influenced by forage type,sample time,and their interactions(P<0.01).Expect the acetate to propionate ratio,all the individual VFA concentrations were significantly higher in rumen of AH-fed animals(P<0.05)than in the animal fed CS.The in-situ degradation of AH in terms of DM,CP and NDF was significantly higher than CS(P<0.05),since AH composed more easily fermentable carbohydrate than CS.When AH was incubated in CS rumen environment,the degradation efficiency of DM,especially NDF and ADF,was accelerated compared with the AH rumen environment(P<0.05),while CP degradation remained unaffected.On the contrary,CS degradation in AH rumen environment was much slower compared to that in CS environment.In addition,during the first 0.5 h of rumen incubation,around 30%of forages was degraded,whereas almost no DM degradation occurred during the 2-4 h of incubation in either rumen environment.The degradation of CS in AH rumen environment was even halted until 8 h after feeding.1.2 Effects on temporal dynamics of microbial colonization on forage particle in situTo obtain a deep understanding the temporal dynamics of rumen microbiota that colonized on and participated in the degradation of forage particles,Illumina sequencing of bacteria 16S rRNA gene was performed.During the first 0.5 h of incubation,the forage was rapidly attached by Prevotella,Succinivibrio,and Succinivibrionaceae UCG-002.After 4 h incubation,the relative abundance of the above-mentioned bacteria decreased progressively,along with increased abundance of Fibrobacter,Treponema 2,Rikenellaceae RC9 gut group,Prevotellaceae NK3B31 group and Ruminococcus 1.Methanogenic Euryarchaeota increased significantly from 0.5 to 4 h when the degradation of forages was halted(P<0.05).The increased methanogenic Euryarchaetoa was possibly related with accumulation of hydrogen as a result of fast degradation of carbohydrate during the first 0.5 h.Significant differences were noted in attached bacterial community structure between the two forages incubated in different rumen environment(P<0.05).The attached rumen community structure was more influenced by rumen environment rather than nutritional components of forages per se.Forages incubated in CS rumen environment was more attached by Butyrivibrio and Fibobacter which were involved in fibrolytic function,while forages incubated in AH rumen environment was more attached by Succiniclasticum,Atopobium,and Desulfovibrio which were involved in soluble carbohydrate and energy metabolism.The above results suggest that the rumen environment formed under AH feeding is not optimal for AH degradation,especially lignocellulose degradation.Our observation indicate that microbiota colonized on forages is highly affected by the rumen environment rather than its chemical components.The rumen environment caused by CS feeing resulted in abundant fibrolytic bacteria such as Fibrobacter and Butyrivibrio that is beneficial for accelerating degradation of AH in the rumen.Overall,dietary intervention using low quality forage may be a method to modulate the rumen environment and then improve forage degradation.2.Rumen fermentation and microbiota influenced by dietary intervention through feeding low quality forageIn order to better understand low quality forage dietary intervention,ten rumen-cannulated Hu sheep were randomly allocated to two dietary treatments(five sheep each)with the same concentration-to-forage ratio and concentration mixture,but different forage sequences:(1)AH to CS and back to CS(ACA sequence);(2)CS to AH and back to AH(CAC sequence),with each treatment lasted for 2 weeks.Except the first period,rumen content samples were collected on d 1,2,4,6,9,and 14 during each treatment.Total collection of feces was conducted for digestibility measurement during the last 5 days in each treatment period.2.1 Effects on nutrients digestibility and rumen fermentationA significantly decrease was observed in the rumen microbial protein concentration on d 6 after dietary transition whether the transition was from AH to CS or from CS to AH,and this was accompanied by an increase in the ammonia nitrogen concentration.However,after transition back to the original forage,the rumen fermentation parameters returned to their initial levels within 2 weeks.In ACA sequence,nutrients digestibility and rumen fermentation parameters were determined to compare the difference between pre-and post-forage intervention.In spite of a slight increase in the digestibility of NDF and as well as VFA concentration,the differences were not significant.2.2 Effects on rumen microbial community structure and temporal adaptationMicrobial variation during intervention was investigated to speculate potentially functional changes in the rumen.We sequenced microbial samples obtained in ACA sequence to monitor the variation and adaptation of rumen microbiota in response to low quality forage intervention.The CS intervention significantly increased the abundance of microorganisms involved in lignocellulose degradation,including Fibrobacter and Treponema(P<0.05).When the diet was switched back to AH,the microbial community did not completely return to pre-intervention status.The abundances of Fibrobacter and Treponema were persistently high(P<0.05),similar to those during CS intervention.Meanwhile,the diversity of the microbial community increased after dietary intervention from AH to CS(P<0.05)and remained significantly higher after transition back to AH compared to those under the origin AH diet(P<0.05).Microbial functional prediction using Tax4Fun revealed that this microbial persistence may enhance the carbohydrate metabolism pathway in the rumen(P<0.05).In addition,we discovered that the increased NH3-N concentration on d 6 after transition was highly correlated with increased relative abundance of Prevotella,which may be linked to the high degradation of feed protein in the rumen.In summary,low quality forage has the potential to accurate lignocellulose degradation in the rumen through modulation of rumen environment and microbiota that colonized on the forage particles.In the meantime,dietary intervention using low quality forage is able to increase the abundance of fibrolytic bacteria and to keep the microbial community into an alternative stability where the increased Fibrobacter and Treponema persisted even after withdrawal of intervention.These knowledges may enlighten the possibility of reprograming the rumen microbial populations and functions using short-term high-fiber intervention to enhance the fibrolytic protentional in the future.In practice,repeated interventions using low-quality forage while feeding animals with high-quality forage may be a promising strategy to enhance the hosts' fibrolytic digestibility and improve animal productivity.