Ensiling Characteristics, in Vitro Rumen Fermentation Profile, Methane Emission and Archaeal and Protozoal Community of Silage Prepared with Alfalfa, Sainfoin and Their Mixture

Feeding ruminant animals with legume silage, rich in non-protein nitrogen, not only results in serious groundwater pollution but also induces a large amount of methane (CH4) releasing into atmospheric environment. Co-ensiling alfalfa with sainfoin containing condensed tannins in five ratios was investigated on silage fermentation, chemical and bacterial community composition, in vitro ruminal fermentation characteristics, CH4 emission and archaeal and protozoal community. Fresh alfalfa and sainfoin were wilted to dry matter concentrations of 381 and 390 g/kg wet weight, respectively, and chopped to 2-3 cm. Chopped alfalfa and sainfoin were combined in proportions of 1:0 (S0, Control), 0.75:0.25 (S25), 0.5:0.5 (S50), 0.25:0.75 (S75) and 0:1 (S100) on a wet weight basis, respectively. Each treatment was prepared in quadruplicate and fermented for 60 days. After 60 days of fermentation, incorporation of sainfoin into alfalfa before ensiling led to an improvement in silage fermentation and inhibited silage proteolysis in a proportion-dependent manner. Excellent fermentation profiles were observed in S50 and S75 silages, in which the nonprotein nitrogen decreased by 11.33% and 13.01%, respectively, compared with S0 silage. As sainfoin proportion increased, the relative abundance of Pediococcus acidilactici linearly decreased (P<0.001) but Lactobacillus pentosus had a quadratic response (P<0.001). In vitro fermentation trial showed that rumen acetate to propionate ratio linearly decreased with more sainfoin (P<0.001). Relative to S0 silage, S50 and S75 silages in vitro rumen CH4 production decreased by 19.16% and 24.25% and dry matter digestibility only dropped by 2.97% and 3.87%, respectively. However, no significant differences between treatments were observed on the major methanogens of Methanobrevibacter (P=0.401) and Entodinium (P=0.858) or Polyplastron (P=0.713) of protozoal community. In conclusion, adding sainfoin into alfalfa prior to ensiling suppressed silage proteolysis and mitigated rumen CH4 emission in a proportion-dependent manner, with a minor negative effect on dry matter digestibility. High-quality legume silage can be produced with S50 and S75 treatments due to the optimal balance of proteolysis inhibition, in vitro rumen CH4 emission and dry matter digestibility. Therefore, co-ensiling of alfalfa with sainfoin could be used as a promising strategy not only to produce high-quality legume silage but also to reduce nitrogen excretion and mitigate rumen CH4 emission from ruminant animals, and further in vivo study is needed to verify this statement.