Ammonia and amino acids modulates enzymes associated with ammonia assimilation pathway by ruminal microbiota in vitro

Abstract

The objective of this study was to evaluate the effects of ammonia and amino acids (AA) supplementation on activities of ruminal enzymes involved in ammonia assimilation. In addition, the temporal changes of ruminal bacterial populations and enzyme activities during in vitro incubation were investigated. Rumen fluid from four ruminally fistulated goats was used in a 3×3 factorial arrangement of treatments with ammonia equivalent to 1, 4, and 15mM ammonium chloride (NH4Cl), and with an added AA mixture (containing 992g casein acid hydrolysate plus 1.4g l-cysteine plus 8.68g l-tryptophan) at 0, 1, and 15.5g/L in the in vitro culture solution. Both ammonia and AA supplementation increased (P<0.01) ammonia–nitrogen and volatile fatty acids concentrations. There was an interaction (P=0.04) between NH4Cl and AA concentrations on the yield of microbial crude protein. The population of total bacteria was dose-dependent with ammonia concentration (P=0.01), but was increased following AA increment (P<0.01). Supplement of AA activated (P<0.01) enzymes of glutamine synthetase (GS), glutamate synthetase (GOGAT), and glutamate dehydrogenase (GDH), but inhibited (P<0.01) enzyme of alanine dehydrogenase (ADH), all of which were important in ammonia assimilation for ruminal microbiota. And all these four enzymes were more active at the later period of process. The GDH activity was significantly associated with the population of Prevotella ruminicola (r=0.66; P<0.01), while the correlations between ADH activity and Fibrobacter succinogenes (r=0.41, P=0.04) or Butyrivibrio fibrisolvens (r=0.45, P=0.02) were not strong. Enzymes of GS, GOGAT and GDH had strong correlations (P<0.01) with yield of MCP. These results suggest that AA supplementation alters fermentation pattern, and stimulates the GS–GOGAT and GDH pathways of ammonia assimilation.