| Methane is an important greenhouse gas produced in the gastrointestinal tract of ruminants.Its emission not only increases the greenhouse effect,but also leads to the loss of total energy of ruminant feed.Previous studies have shown that the host and diet are important factors affecting the methane production of ruminants,and the methane production of cervids is lower than that of bovine ruminants such as cattle and sheep,among which roe deer is the most representative.In addition,ruminant gastrointestinal microbes play a decisive role in methane production.Analyzing the microbiological mechanism of roe deer low methane production can provide a theoretical basis for reducing methane production and improving feed utilization efficiency of ruminant livestock.Therefore,this study took roe deer and goats as the research objects,fed the same diet,and compared the gas production characteristics of roe deer and goats,as well as the community structure of bacteria and methanogens in the digestive tract,through in vivo and in vitro experiments.This research mainly includes the following contents:Experiment 1: Comparative study on gas production,digestibility and Energy Metabolism of roe deer and goatIn this experiment,four 2-year-old,healthy male roe deer(BW = 31.4 ± 0.31 kg)and goats(BW = 31.4 ± 0.30 kg)were selected as research objects,fed with the same total mixed diet(TMR,concentrate: roughage = 30:70,dry matter(DM)basis),the same amount of feeding twice a day,the test lasted 24 days,of which the first 21 days were the adaptation period,and the last 3 days were the formal test period.During the official test phase,the gas production of roe deer and goats was monitored through the breathing chamber,and the apparent digestibility of fecal nutrients,volatile fatty acids(volatile fatty acids,VFAs)concentration and energy metabolism were measured.The test results showed that the methane production of roe deer was significantly lower than that of goats(P < 0.05),while the difference in carbon dioxide production was not significant(P > 0.05).The dry matter,crude protein(CP)and neutral detergent fiber(NDF)digestibility of roe deer feces were significantly lower than those of goats(P < 0.05),and the methane production of digestible NDF,DM and CP was significantly lower than that of goats(P < 0.01).The fecal energy(FE)and metabolizable energy(ME)to digestible energy(DE)ratios of roe deer feces were significantly higher than those of goats(P <0.05).The results of fecal VFAs showed that the mole percentage of propionate in roe deer feces was significantly higher than that of goats(P < 0.05),while the mole percentages of valeric acid,isobutyric acid,and isovaleric acid were significantly lower than those of goats(P < 0.05).The above results show that the methane production and apparent digestibility of DM,CP and NDF roe deer were significantly lower than that of goat.Experiment 2: Comparative study on the bacterial community structure of roe deer and goat fecesUsing the feces obtained in the above experiments as objects,the microbial genomic DNA was extracted,the bacterial 16 S r RNA gene V3-V4 region was amplified with 341F-806 R primers,sequenced by Illumina Mi Seq,the microbial composition was analyzed by QIIME1.9.1,and the microbial function was predicted based on PICRUSt.Analysis and comparison of roe deer and goat fecal bacterial community structure.The results showed that the dominant bacterial phyla in roe deer and goat feces were Firmicutes and Bacteroidetes,and the dominant bacterial genera were Oscillospiraceae UCG 005,Rikenellaceae RC9,Oscillospiraceae UCG010 and Christensenellaceae R7.α-diversity analysis showed that the number of OTUs and Chao1 index of roe deer fecal bacteria were significantly higher than those of goats(P < 0.01).Principal coordinates analysis(PCo A)showed that there was a significant difference in the fecal bacterial community structure between roe deer and goat(P < 0.05).The relative abundances of Bifidobacterium spp.,Ruminococcus spp.,Succinivibrio spp.,Treponema spp.,and Prevotella spp.in feces of roe deer were significantly higher than those of goats(P < 0.05),while Bacteroides spp.,Alistipes spp.,Blautia spp.,and Lachnospiraceae members(Lachnospiraceae unclassified,Lachnospiraceae uncultured,Lachnospiraceae FCS020)was significantly lower than that of goats(P < 0.05).The comparative analysis of microbial predictive functions showed that the relative abundance of propionate and butyrate metabolic pathways in roe deer fecal microorganisms was significantly higher than that in goats(P < 0.05).The above results indicated that the fecal bacterial community structure of roe deer was significantly different from that of goats,leading to differences in the production mechanism of VFAs.Experiment 3: Comparative study on the community structure of roe deer and goat fecal methanogensBased on the roe deer and goat fecal microbial genomic DNA,the whole intestinal sequence of the 16 S r RNA gene of Methanogen was amplified with Met86F-Met1340 R primers,sequenced by Pac Bio(Pacific Biosciences),and the microbial composition was analyzed by QIIME1.9.1.Analysis and comparison of the community structure of roe deer and goat fecal methanogens.The results showed that the dominant methanogens phylum in roe deer and goat feces were Halobacterota and Euryarchaeota,respectively,and the dominant methanogens were Methanocorpusculum spp.and Methanobrevibacter spp.,respectively.Methanocorpusculum uncultured and Methanobrevibacter unclassified 2 were the dominant methane species in roe deer feces,while Methanobrevibacter unclassified 2 and Methanobrevibacter sp.YE350 were the dominant methane species in goat feces.α-diversity analysis showed that the Simpson index of roe deer fecal methanogens was significantly higher than that of goats(P < 0.01).The results of PCo A showed that there were significant differences in the community structure of fecal methanogens between roe deer and goats(P < 0.05).The relative abundance of roe deer feces Methanocorpusculum uncultured and Methanosphaera unclassified 4 was significantly higher than that of goats,while the relative abundances of Methanobrevibacter millerae,Methanobrevibacter sp.YE 315 and Methanobrevibacter ruminantium M1 were significantly lower than those of goats.Methane production in roe deer feces was positively correlated with Treponema spp.and negatively correlated with Bifidobacterium spp.,while NDF digestibility was positively correlated with Christensenellaceae R7 and Methanobrevibacter unclassified 2and negatively correlated with Succinivibrio spp.,and Methanocorpusculum uncultured.Methanobrevibacter ruminantium M1 of goat feces was positively correlated with the genera Blautia spp.and Treponema spp.,negatively correlated with Ruminococcus spp.,Succinivibrio spp.was negatively correlated with DM and NDF digestibility.The above results indicated that the community structure of roe deer fecal methanogens and their correlation network with bacteria were significantly different from those of goats.Experiment 4: Comparative study on rumen liquid fermentation of roe deer and goatThis experiment adopts a single-factor horizontal test design,and is divided into roe deer group and goat group.Each group has 6 time points,and each time point has 4 repetitions.and24 h of rumen liquid for external fermentation.The results showed that the total gas production,total VFAs,acetic acid,propionic acid,butyric acid and valeric acid molar concentration of roe deer rumen fermentation broth increased with the increase of fermentation time and were significantly lower than those of goats(P < 0.05).The p H of roe deer fermentation broth decreased with the increase of fermentation time,but it was significantly higher than that of goats(P < 0.05),while the degradation rate of DM was higher than that of goats with the increase of fermentation time,and there was a significant difference between 2 h and 4 h(P <0.05).The above results show that the fermentation rate of roe deer is different from that of goat,and the fermentation ability of roe deer is lower than that of goat.In summary,this study found that under the same diet,the methane production of roe deer is significantly lower than that of goats,the composition of fecal bacteria and methanogens in roe deer is significantly different from that of goats,and the metabolic pattern of fecal propionate in roe deer is significantly different from that of goats. |
