Effects Of Additives On Quality Regulation And Microbial Diversity Of Alfalfa Mixed Silage

Alfalfa(Medicago sativa)is a perennial high-quality leguminous forage widely planted in the world.With the increasing demand for forage in herbivorous animal husbandry,the planting area of alfalfa in southern China has been expanding in recent years.Mixed silage of fresh alfalfa with rice straw or other agricultural and sideline products can solve the problem of safe storage of alfalfa.Additives are the effective way to improve the fermentation and nutritional quality of silage.In this study,lactic acid bacteria,cellulase and short-chain fatty acid additives were added to the mixed silage of alfalfa.Through dynamic analysis of the fermentation characteristics and chemical components of the mixed silage,and the comprehensive evaluation of in vitro rumen degradation rate,rumen fermentation parameters and gas production,combined with the dynamic change analysis of bacterial flora structure succession and diversity,this research aims to reveal the mechanism of silage fermentation with different additive formulas,provide valuable biological information for the succession of microbial communities in alfalfa mixed silage system,and establish a suitable southern silage system.The main research conclusions are as follows:(1)The objective 1 of this study was to determine the effect of pre-fermented juice(J),Lactobacillus plantarum(LP),and L.buchneri(LB)of alfalfa mixed silage.Four lactic acid bacteria(LAB)species from three genera were cultured from the pre-fermented juice,with W.cibaria being dominant.Adding LP+LB increased abundance of L.plantarum,L.paralimentarius and L.nodensis,resulting in the lowest p H.Pre-fermented juice enriched W.cibaria,L.sakei,L.parabrevis,Pseudomonas putida,and Stenotrophomonas maltophilia,mainly enhanced accumulation of acetic acid(AA)and lactic acid(LA),and decreased p H,crude protein(CP)losses,ammonia(AN),and hemi-cellulose(HC)contents.In addition,various inoculants had no obvious effect on the improvement of in vitro digestibility.In conclusion,inoculations altered bacterial succession in silage;LB and pre-fermented juice enhanced ensiling by promoting p H reductions,enhancing concentrations of LA and AA,and extending aerobic stability more than 16 d.(2)The objective 2 of this study was to determine effects of potassium diformate(PD),sodium diacetate(SD)and calcium propionate(CAP)of alfalfa mixed silage.The positive correlation between PD and L.plantarum supported the lowest p H,butyric acid(BA),AN,neutral detergent fiber(NDF)and acid detergent fiber(ADF),and HC content,and high water-soluble carbohydrates(WSC)and CP content in PD silage.In addition,SD and CAP enriched the abundance of L.parabrevis and mainly increased LA and AA.CAP increased abundance of L.acetotolerans after 45 d of ensiling with more LA and AA than other treatments.In conclusion,the succession of the bacterial community of mixed silage was modulated by the three fatty acid salts;furthermore,PD and CAP further improved fermentation quality by accelerating the decrease in p H and the increase in LA.The three fatty acid salts have significant effects on improving in vitro dry matter digestibility(IVDMD)and aerobic stability.(3)The objective 3 of this study was to determine the effect of alfalfa mixed silage.The addition of three enzymes enriched the abundance of Lactobacillus and Weissella and increased LA,AA,WSC,DM,CP contents,improved IVDMD,whereas inhibited the growth of undesirable microbes,resulted in lower p H,AN,BA,NDF contents.Adding X and EX increased the abundance of L.paralimentarius and L.parabrevis,enhanced accumulation of AA,and reduced HC.The E group had the highest abundance of W.cibaria.EX-treated silage enriched abundance of Bacillus velezensis,had the potential to improve increased IVDMD,total VFA production,and gas production during in vitro fermentation.Aerobic stability was prolonged(>16 d)by E,X or EX-treated silages.In conclusion,the addition of three enzymes altered bacterial succession,improved fermentation quality;furthermore,X and EX enhanced ensiling by promoting p H reductions and enhancing concentrations of AA.For in vitro rumen degradability,EX treated silage had the highest DM degradability,total VFA production,and lowest p H.(4)By studying the effect of J broth with LB,EX,PD and CAP on the mixed silage of alfalfa,it was found that the compound additive reduced the effect of mixed silage.The p H value and AN content increased the WSC content and inhibited the growth of Clostridia.J+X,J+EX and J+PD treatments increased the relative abundances of Stenotrophomonas maltophilia and Pseudomonas putida,and significantly decreased bacterial diversity,p H and AN content,while J+X and J+EX treatments group significantly increased the AA content.The J+CAP treatment group had the highest LA and AA content.The J+EX treatment group significantly reduced NDF,ADF and hemicellulose content,and increased IVDMD and IVNDFD.The J+LB treatment group increased the relative abundance of L.buchneri and L.acetotolerans,as well as increased IVDMD and IVNDFD.J+EX,J+PD and J+CAP treatment groups significantly improved aerobic stability(>16 d).Based on the effects of various additives on the fermentation quality,chemical composition,in vitro digestibility and aerobic stability of alfalfa mixed silage,PD,EX and J+EX were finally recommended as production silage additives.The above three additives can not only improve fermentation quality,IVDMD and IVNDFD,and aerobic stability,but also reduce the loss of DM,CP and WSC.