| The rumen is important for ruminants to utilize fiber.Rumen micro-ecosystem,as one of the most complex and valuable ecosystems in the world,has been the object of in-depth research in nutrition and microbiology.Domestic and foreign research results showed that rumen anaerobic fungi play an important role in fiber degradation and rumen internal environment homeostasis,which is closely related to production efficiency of ruminants.Therefore,it is of great theoretical and practical significance to screen efficient rumen anaerobic fungi and study their effects on fiber degradation and rumen environment.This paper was divided into three experiments by in vitro.In experiment 1,Hungate rolling tube method was used to purify,screen and enrich rumen anaerobic fungi based on the degradation rate of DM.Then,A two-factor completely randomized trial design was used,including 4 strains（A,B,C,D）and two substrates,rice straw（G）and corn straw（J）,with a total of 8 treatments and 3 replicates per treatment.The incubation time was 48 h,and the degradation rate of DM was determined.Four strains of anaerobic fungi A,B,C and D were isolated and purified from rumen fluid of Holstein dairy cows.When substrate was G,the degradation rate of Bacteria C was the highest,which was significantly higher than that of bacteria B（P<0.05）,but there was no significant difference with that of bacteria A and D（P>0.05）.When substrate was J,the dry matter degradation rate of Bacteria C and D was significantly higher than that of bacteria A and B（P<0.05）,but there was no significant difference between bacteria C and D,and between bacteria A and B（P>0.05）.Based on the above results,bacteria C and D were selected for subsequent tests.The 28S r RNA gene sequences of Bacteria C and D were analyzed and compared,and the two strains were identified as Phialemoniopsis SP.and Cladosporium sp.（later abbreviated as Fungi P and C）.Experiment 2 was conducted on the basis of Experiment 1.Two anaerobic fungi selected were used,and 6 inoculum doses and 4 kinds of roughage were selected to combination in vitro in order to obtain the inoculation dose that maximized the degradation rate of DM and NDF of different feeds.The 4 types of roughage were silage corn（Q）,corn stalks（J）,alfalfa（M）and Leymus chinensis（Y）.The inoculation doses were 1%,3%,4%,5%,and 7%.No fungi served as the control group.The incubation time was 48 hours,and96 treatments were performed to determine the degradation rate of DM and NDF.The results showed that,adding different doses of Fungi P,there was no significant difference in the degradation rate of NDF among all groups except NQ group（P>0.05）,and the 4%dose in NQ group was significantly higher than 3%dose（P<0.05）.The dose and feed source of different groups had significant differences in DM degradation rate（P<0.05）.The comprehensive experimental results showed that the addition of Fungi P inhibited the degradation of straw and Leymus chinensis,but promoted the degradation of silage and alfalfa.The silage and alfalfa were selected for the following experiment,and the dose of silage was 1%and that of alfalfa was 3%.Adding different doses of Fungi C,the results showed that there was no significant difference in the degradation rate of NDF among all groups except NM group（P>0.05）,and the 5%dose group in NM group was significantly higher than 7%group（P<0.05）.The results indicated that the degradation rate of NDF did not increase with the increase of dose.Adding different doses of Fungi C had significant difference in DM degradation rate（P<0.05）,1%and 3%dose groups in DQ group were significantly higher than those in other groups（P<0.05）,7%dose group in DJ group was significantly higher than that in 3%and4%groups（P<0.05）,1%dose group in DY group was significantly higher than that in 7%group（P<0.05）.Comprehensive analysis results showed that,DQ group was significantly higher than DM group,and extremely significantly higher than DJ and DY groups.Therefore,silage and alfalfa were also selected for further study,and the dose of both was 1%.In experiment 3,the effects of exogenous anaerobic fungi on rumen fermentation parameters were studied at the optimal inoculation dose.According to the results of experiment 2,different feeds and strains were used as influencing factors to obtain 4combinations of Silage×Fungi P（QP）,alfalfa×Fungi P（MP）,silage×Fungi C（QC）and alfalfa×Fungi C（MC）.Each group was divided into three treatments.There were experimental groups（QP,MP,QC,MC）,positive control group and negative control group,and each group had 3 replicates per treatment.The culture time was 0 h,1 h,2 h,6 h,12 h,24 h and 48 h,and p H,NH3-N concentration,BCP concentration and VFA concentration were determined.The results showed that,for p H value,QP and QC groups were significantly different（P<0.05）,positive control group was significantly higher than negative control group（P<0.05）,extremely significantly higher than experimental group（P<0.01）,negative control group was significantly higher than experimental group（P<0.05）,There was no significant difference among MP and MC groups（P>0.05）.For NH3-N concentration,there was no significant difference in all treatment groups except QP（P>0.05）,and the positive control group in QP group was significantly higher than the other two groups（P<0.05）,and there was no significant difference between the latter two groups（P>0.05）.For BCP concentration,whole corn silage as substrate,Fungi P and C in experimental groups were significantly higher than those in other two groups（P<0.05）;With alfalfa as substrate,the concentration of Fungi P in the experimental group was significantly higher than that in the positive control group（P<0.05）,but there was no significant difference to negative control group（P>0.05）,while the concentration of Fungi C in the negative control group was the highest,which was significantly higher than the other two groups（P<0.05）.It indicated that Fungi P could significantly increase the concentration of BCP,while Fungi C was affected by different substrates.For VFA concentration,there was no significant difference in acetic acid concentration in QP and QC groups（P>0.05）,the negative control group in MP group was significantly higher than that in positive control group and experimental group（P<0.05）,the negative control group in MC group was significantly higher than that in experimental group（P<0.05）,and there was no significant difference between positive control group and the other two groups（P>0.05）.Propionic acid concentration in QP,MP and MC groups had no significant difference（P>0.05）,and that in QC group was significantly lower than that in negative and positive control groups（P<0.05）.There were no significant differences in butyric acid concentration,TVFA concentration and ratio of b to C in QP,QC,MP and MC groups（P>0.05）.For the influence of time,there were significant differences in all indexes of each combination（P<0.05）,indicating that time had significant influence.In summary,under the conditions of this experiment,the DM degradation rate of roughage by Fungi P and C purified from the rumen juice of dairy cows varied with different substrates,which could promote the DM degradation rate of silage and alfalfa;Under the conditions,the best inoculation dose of Fungi P was 1%for silage,alfalfa was 3%,and the inoculation dose of Fungi C was 1%for both feeds.The result of comprehensive rumen environmental indicators showed that Fungi P had a greater impact,impact of Fungi C was less,and the concentration of BCP canould be significantly increased.It was expected that additives of improving fiber degradation will be developed to serve the production of ruminants. |
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