Effects Of Different Silage Methods On Alfalfa Silage Quality And Microbial Diversity

Object:In this study,fresh alfalfa grass was used as the raw material.Silage methods include single alfalfa silage,corn flour and alfalfa mixed silage,pine needles and alfalfa mixed silage.The differences in silage quality and microbial diversity of the three silage methods were analyzed and compared,and the effects of corn flour and alfalfa mixed silage,pine needles and alfalfa mixed silage on rumen fermentation and microbial structure of lambs were explored.So as to provide reference for the production and application of pine needles and alfalfa mixed silage.Methods:This paper was divided into three experiments.The specific methods were as follows:Experiment 1:Alfalfa silage was used as the control group（CON group）.According to the fresh weight of alfalfa grass,corn flour and alfalfa were mixed at a ratio of 1:9（CAMS group）,pine needles and alfalfa were mixed at a ratio of 3:7（PAMS group）.Silage tank sealed.Each processing has three replicates.And samples were taken at the 0,1,3,5,7,15,30,45 and 60 days of silage fermentation.The sensory quality,nutrient components,fermentation quality,mycotoxin content and aerobic stability of silage were analyzed.The changes of the main antibacterial components in pine needles were analyzed by gas chromatography-mass spectrometry.Experiment 2:On the basis of experiment 1,the changes in the number of lactobacillus,yeast,mold,aerobic bacteria and Escherichia coli were determined by the coating plate method.The effects of different treatments on microbial diversity of alfalfa silage were analyzed by high-throughput sequencing.Experiment 3:Fifty male Kazakh lambs were divided into 5 groups by similar body weight.Lambs were fed diets with a 50:50 concentrate to roughage ratio,mixed silage was 35%in the diets.The diet silage was composed of pine needles and alfalfa mixed silage,corn flour and alfalfa mixed silage.The percentage of pine needles and alfalfa mixed silage in silage were 0（groupⅠ）,1/6（groupⅡ）,1/3（groupⅢ）,2/3（groupⅥ）,1（groupⅤ）,respectively.On the last day of the trial period,four fattening lambs in each group were selected to collect rumen fluid.The effects of two kinds of alfalfa mixed silage on rumen fermentation and microbial community structure of fattening lambs were compared.Results:Experiment 1:1)Loss rates of DM,CP and WSC in pine needles and alfalfa mixed silage were significantly lower than those in corn flour and alfalfa mixed silage（P<0.05）.Loss rates of DM,CP and WSC in mixed silage of corn flour and alfalfa were significantly lower than those in alfalfa silage alone（P<0.05）.2)At the end of silage,the NH₃-N/TN in the mixed silage of pine needles and alfalfa was significantly lower than that in the single alfalfa silage and the mixed silage of corn flour and alfalfa（P<0.05）.And the contents of acetic acid,propionic acid and butyric acid in the mixed silage of pine needles and alfalfa were significantly lower than those in the single alfalfa silage and the mixed silage of corn flour and alfalfa（P<0.05）.3)The contents of AFT,ZEN and T-2 toxins in the mixed silage of pine needles and alfalfa were significantly lower than those in the single alfalfa silage and the mixed silage of corn flour and alfalfa（P<0.05）.4)Aerobic stability of pine needles and alfalfa mixed silage was significantly higher than that of the corn flour and alfalfa mixed silage（P<0.05）.Aerobic stability of mixed silage of corn flour and alfalfa was significantly higher than that of alfalfa alone（P<0.05）.5)The relative contents of monoterpene,sesquiterpene and total terpenes in the mixed silage of pine needles and alfalfa decreased with the extension of silage time,while the content of oxygenated terpene increased first and then decreased.Experiment 2:1)From the 7th day of silage,the number of lactobacillus in the mixed silage of corn flour and alfalfa,and the mixed silage of pine needles and alfalfa was significantly higher than that in single alfalfa silage（P<0.05）.The number of yeasts and aerobic bacteria in the mixed silage of pine needles and alfalfa were significantly lower than those in the single alfalfa silage and the mixed silage of corn flour and alfalfa（P<0.05）.At the end of silage,molds were not detected in the single alfalfa silage and the mixed silage of corn flour and alfalfa.And molds and Escherichia coli were not detected in the mixed silage of pine needles and alfalfa.2)The richness of bacteria and fungal flora in each treatment group showed a downward trend with the extension of silage time.At the end of silage,the relative abundances of Firmicutes and Lactobacillus in the mixed silage of pine needles and alfalfa were higher than those of alfalfa silage alone and mixed silage of corn flour and alfalfa.The relative abundances of Ascomycota and Basidiomycota in the mixed silage of pine needles and alfalfa was lower than those of alfalfa silage alone and mixed silage of corn flour and alfalfa.Experiment 3:1)The rumen p H was significantly higher in groupⅤthan that in groupⅠ（P<0.05）.The concentration of acetic acid and acetic acid/propionic acid were significantly higher in groupⅤthan those in groupⅠ（P<0.05）.The concentration of ammonia nitrogen was significantly lower in groupⅤthan that in groupsⅠandⅡ（P<0.05）.The concentrations of propionic acid and butyric acid were significantly lower in groupⅤthan those in groupⅠ（P<0.05）.2)The relative abundance of Firmicutes was significantly higher in groupsⅠandⅡthan that in groupsⅣandⅤ（P<0.05）.The relative abundance of Prevotella was significantly higher in groupⅠthan that in groupⅤ（P<0.05）.The relative abundances of Succiniclasticum and Ruminococcus were significantly higher in groupsⅠ,ⅡandⅢthan those in groupsⅣandⅤ（P<0.05）.Conclusion:Compared with alfalfa silage alone,corn flour and alfalfa mixed silage,pine needles and alfalfa mixed silage could improve the quality of alfalfa silage to varying degrees.The effect of pine needles and alfalfa mixed silage was the best.The contents of monoterpene,sesquiterpene and total terpenes in the mixed silage of pine needles and alfalfa decreased with the extension of silage time,while the content of oxygenated terpene increased first and then decreased.Mixed silage of pine needles and alfalfa significantly reduced the number of yeasts,aerobic bacteria,Escherichia coli and fungal community abundance.Under the conditions of this experiment,the mixed silage of pine needles and alfalfa completely replaced the mixed silage of corn flour and alfalfa could significantly reduce the concentration of ammonia nitrogen and propionic acid in the rumen of lambs and reduce the abundance of rumen flora.