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Phyto-sources possessing different secondary metabolites are under investigation for the mitigation of enteric methane emission from livestock. Alfalfa (Medicago sativa) contains considerable saponin which is known for methane reduction through anti-protozoal action. Therefore, this study was undertaken to ascertain the effect of saponin containing alfalfa fodder (Medicago sativa; second cut) on enteric methane emission in Murrah buffaloes quantified using sulfur hexafluoride (SF6 ) technique. Twelve male Murrah buffalo calves were randomly divided into two groups of six animals each. Buffalo calves in control group were fed on wheat straw and concentrate based diet (R: C, 60:40), while animals in test group were supplemented with saponin containing alfalfa fodder (second cut, 30%) replacing wheat straw on w/w basis. Effect of alfalfa fodder supplementation on rumen fermentation characteristics, archaeal and protozoal population were also studied. Enteric methane emission in control and test group buffaloes was reported as 78.09 and 61.39 g/d, respectively. In this study, about 21% reduction in enteric methane emission was achieved on the feeding of saponin containing alfalfa fodder at 30% level of the diet. However, dry matter intake, pH, ammonia nitrogen and total volatile fatty acid production did not differ (p>0.05) among the groups. A significant (p<0.05) decrease in acetate production was also with concomitant increase in propionate production. Results revealed a nonsignificant change in archaeal population, while protozoal population were adversely affected and about 20% less numbers were observed in test group. From the study it may be concluded that saponin from natural feed resources like alfalfa fodder at a level of 6.0 g/kg DM can be used for the significant enteric methane reduction.