Abstract: Methods of in vitro culture technique and orthogonal experimental design were using in this experiment to identify the effects of branched chain amino acids (BCAAs,valine,leucine and isoleucine) on rumen microbial fermentation in vitro based on different concentrate to forage ratio (CFR) of 20:80,40:60 and 60:40,and the best combination of BCAAs on each CFR was gained after analysis of the combination effects of BCAAs.The results showed that the final pH,NDF degradability (NDFD) and acetate/propionate were not affected by BCAAs addition (P>0.05).However,DM degradability (DMD) was significantly different in various combinations.When the CFR was 20:80,the three amino acids showed a combination significant effect on the NH₃-N,VFA and TVFA (P<0.05).Along with the increase of valine levels,the concentrations of isobutyric acid and valeric acid were significantly increased (P<0.01),isovaleric acid was affected by isoleucine and leucine (P<0.05).When the CFR was 40:60,the concentration of isobutyric acid increased with the increase of valine supplementation level (P<0.01).Isovaleric acid was affected by three kinds of BCAAs (P<0.05).When the CFR was 60:40,BCAAs mainly affected the concentrations of isobutyric acid, valeric acid and isovaleric acid.Isobutyric acid was mainly affected by valine (P<0.01),isovaleric acid was affected by leucine and isoleucine (P<0.01),and valeric acid was affected by valine and isoleucine (P<0.05).Based on the concentrations of isobutyric acid,valeric acid and isovaleric acid,the three BCAAs showed obvious combination effects (P<0.05).In conclusion,taking DMD as the index,when the CFR was 20:80,the best supplementation levels of valine,leucine and isoleucine were 0.67, 2.00 and 1.33 mmol/L,respectively, when the CFR was 40:60 or 60:40,it's not necessarily to add these BCAAs.

Key words: branched chain amino acids; fermentation in vitro; concentrate to forage ratio; rumen microbes; combined effect