Effects of lactic acid bacteria and grape seed tannin on enhancing intestinal supply of essential amino acids from alfalfa silage through improved preservation and rumen bypass in a multi-stage in vitro system

The limited bioavailability of essential amino acids (EAAs) in alfalfa silage, caused by proteolysis during ensiling and extensive ruminal degradation, restricts its protein nutritional value for ruminants. This study evaluated the individual and combined effects of lactic acid bacteria (LAB) and grape seed tannins (G) on the dynamic fate of essential amino acids (EAAs) throughout ensiling, ruminal fermentation, and intestinal digestion. LAB treatment accelerated acidification and improved EAA preservation during ensiling but, importantly, increased ruminal degradation of most EAAs. In contrast, tannins alone reduced ruminal degradation of key EAAs. Compared with the control, the combined LG treatment enhanced the retention of EAAs and reduced NH 3 -N concentration by 25.5% during ensiling. In the subsequent in vitro rumen fermentation, this treatment also suppressed the growth of proteolytic bacteria ( Butyrivibrio fibrisolvens and Prevotella ruminicola ) and lowered branched-chain VFA concentrations. This resulted in the lowest ruminal degradation rates for most EAAs and the highest intestinal digestibility. In conclusion, the combination of LAB and grape seed tannins optimizes the entire protein utilisation pathway from silage preservation to rumen bypass and intestinal availability, effectively enhancing post-ruminal supply of digestible EAAs. This strategy offers a practical approach to improve protein efficiency in ruminant production, pending in vivo validation.