Screening Of Enterococcus Faecium And Its Effects On Nutrient Digestion,rumen Fermentation And Immunity In Sheep

In this experiment,the optimal addition level of Enterococcus faecium in sheep was selected by in vitro,and Bacillus subtilis was used for comparison,and added into sheep feed to study the effects on nutrient digestion,rumen fermentation and immunity of sheep.The effects of adding single bacteria and compound probiotics were compared,which provided scientific basis for the application of Enterococcus faecium in ruminant feed additives and ideas for healthy breeding.In this study,in vitro and in vivo were carried out to select the suitable adding activity of Enterococcus faecalis in sheep rumen fluid.The feeding experiments with 15 days of pre-feeding period and 60days of positive feeding period,0～30 days（Phase I）and 31～60 days（Phase II）of positive feeding period.Rumen fluid was collected at different time points from 1 to 3 days of each period and rumen fermentation indexes were analyzed.Digestion trial were conducted on the 5th to 12th day of each period to determine the apparent digestibility of nutrients,and the sheep production performance was measured on the 30th day of each period.Rumen fluid was collected before the 30th and 60th day of morning feeding,and the rumen microflora was analyzed by 16S r DNA sequencing.Blood samples were collected from jugular vein before the 0th,15th,30th,45th and 60th day of morning feeding to determine the serum immune indexes.And the data are sorted and analyzed.It is intended to provide scientific basis for exploring the application of Enterococcus faecalis and Bacillus subtilis in sheep feed additives.This experiment includes the following five aspects:Test 1:Screening the optimal addition level of Enterococcus faecalis in vitro.The purpose of this experiment was to study the effects of Enterococcus faecalis with different addition on rumen fermentation parameters of sheep in vitro,and to explore the optimal level of supplementation in sheep.In this experiment,the method of gas production in vitro was adopted,and the control group,group 1,group 2 and group 3 were set up.The viable count of Enterococcus faecalis in each group was 0,1×10¹² CFU/m L,1×10¹¹ CFU/m L and 1×10¹⁰ CFU/m L,respectively.The gas production was measured after incubation for 3,6,12,24 and 48 hours,and the p H was measured at 24 and 48 hours.The results showed that the gas production of each treatment group was significantly higher than that of the control group（P<0.05）.The p H of each group decreased with the increase of time;The dry matter degradation rate gradually increased with the increase of time,and the dry matter degradation rate of group 3 was significantly higher than that of control group at 24 hours（P<0.05）.The concentration of NH₃-N in each treatment group increased with the decrease of Enterococcus faecalis activity,and the concentration of NH₃-N in group 3 was significantly higher than that in control group at 24 h（P<0.05）.Adding Enterococcus faecalis significantly increased the concentrations of acetic acid,propionic acid and TVFA（P<0.05）.This experiment suggested that the optimal added level of Enterococcus faecalis in sheep was 1×10¹⁰CFU/m L.Test 2:Effects of different probiotics on production performance and digestibility of nutrients in sheep.In this experiment,24 adult sheep weighing（32.7±4.7）kg and fitted with fistula were selected.They were CK group（10 m L fermentation broth）,S group(10 m L 1×10¹⁰ CFU/m L fermentation broth of Enterococcus faecalis),K group(10 m L 1×10¹⁰ CFU/m L fermentation broth of Bacillus subtilis)and F group(5 m L 1×10¹⁰ CFU/m L fermentation broth of Enterococcus faecalis+5 m L1×10¹⁰ CFU/m L Enterococcus faecalis fermentation broth).Digestion test was carried out on the5th to 12th day of each feeding period,and nutrient digestibility was determined.The daily gain of experimental animals was measured on the 30th day of each feeding period,and F/G was calculated.The results showed that at stage I,ADG of group S and group K was higher than that of group CK（P>0.05）,F/G of group F was the highest,EE digestibility of group K and group F were significantly lower than those of group CK（P<0.05）,F/G of group S was the highest,and ADF digestibility of group S was significantly higher than that of group CK（P<0.05）.To sum up,adding Enterococcus faecalis for 60 days is more conducive to the digestion of nutrients in sheep.Test 3:Effects of different probiotics on rumen fermentation parameters of sheepExperimental animals and groups are the same as those in Experiment 2.Rumen fluid was continuously collected for 0 h,2 h,4 h,6 h and 8 h from the 1st to 3rd day of each period,and p H,NH₃-N and VFA were determined.The results showed that in the first stage,There was no significant difference in p H,NH₃-N and VFA content between the treatment groups and the CK group（P>0.05）.At stage II,acetic acid in group K was significantly lower than that in group S and group F（P<0.05）,propionic acid in group K was significantly lower than that in group CK and group S（P<0.05）,TVFA concentration in group K was significantly lower than that in other groups（P<0.05）,and ethylene-propylene ratio in group F was significantly higher than that in other groups（P<0.05）.To sum up,from the rumen fermentation index,the effect of compound probiotics is better than that of Enterococcus faecium and Bacillus subtilis.Test 4:Effects of different probiotics on rumen microflora of sheepExperiment and grouping are the same as experiment 2.Rumen fluid was collected within 0～1 hour before feeding on the 30th day of each period,and protozoa count and 16S r DNA sequencing were carried out.The results showed that there was no significant difference in the number of protozoa between each treatment group and CK（P<0.05）.At stage I,there was no significant difference in the relative abundance of dominant bacteria and genera between the treatment groups and the CK group（P<0.05）.At stage II,the relative abundance of unclassified genus-F082 in group S was significantly lower than that in group CK（P<0.05）,the relative abundance of Verrucomicrobia in group K and F was significantly lower than that in group CK（P<0.05）.To sum up,addition of Enterococcus faecium,Bacillus subtilis and their composite microbial agents for 60 d could affect the relative abundance of some phyla or genera of rumen microorganisms in sheep.Test 5:Effects of different probiotics on serum immune indexes of sheepExperiment and grouping are the same as experiment 2.Blood samples were collected at 0,15,30,45 and 60 days during the feeding period for the determination of serum immune indexes.The results showed that at 0 d,the IL-2,TNF-αand Ig A contents in group S and group K were significantly higher than those in other groups（P<0.05）,and the Ig G content in group CK was significantly lower than that in other groups（P<0.05）.At 15 d,IL-2 in group F was significantly higher than that in group CK（P<0.05）,IL-6 in group CK was significantly higher than that in group S and group K（P<0.05）,Ig A,Ig G,Ig M and TNF-αin group S and TNF-αin group K were significantly higher than those in other groups（P<0.05）;at 30 d,IL-2 in CK group,Ig A in S group and IL-2 and Ig M in F group were significantly higher than those in other groups（P<0.05）,and IL-6 in S group and K group was significantly lower than that in CK group（P<0.05）;at 45 d,IL-2,IL-6,Ig A in group S,IL-6,Ig A and Ig M in group F were significantly higher than those in other groups（P<0.05）,TNF-αin group CK was significantly higher than that in group K and group F（P<0.05）;at 60 d,IL-2 and IL-6 in CK group,Ig A,Ig M,TNF-αin S group and Ig M in F group were significantly higher than those in other groups（P<0.05）.To sum up,adding Enterococcus faecalis and Bacillus subtilis can increase the concentration of immunoglobulin and cytokine in sheep,and the effect of single microbial agent is better than that of compound probiotics.Conclusion:Adding Enterococcus faecalis can promote nutrient digestion of sheep,improve sheep’s immune indexes and regulate the phylum or genus of sheep rumen microflora,the addition of complex microbial agents could affect the rumen fermentation type,and the effect of 60 d of Enterococcus faecalis is better.