Comparative evaluation of methods for ammonia (NH

Ammonia (NH3) is main product of ruminai protein degradation and serves as an indicator of nitrogen availability for microbial protein synthesis. Several analytical methods exist to quantify NH3 concentration, but their reliability varies with the analytical approach and experimental setup. This study aimed to compare four NH3 determination methods: Conway microdiffusion, Ion Selective Electrode (ISE), Indophenol, and Nessler, for evaluating in vitro fermentation of legumes and concentrate samples. Supernatants from legumes (n=7) and concentrates (n=7) after 4 h incubation, with four replicates each, were analyzed using these methods. Data were subjected to a 2×4 factorial randomized block design followed by Duncan test. Correlation and regression analyses were conducted between the Conway method and other methods across legume, concentrate, and mixed samples. The ISE method produced the highest NH3 concentration (93.63 mM), while the Indophenol and Nessler methods yielded values closer to Conway. Significant correlations (P<0.05) were observed between Conway and other methods in mixed samples, with coefficients ranging from 0.5327 to 0.6663, but depended on substrate type. In conclusion, Indophenol and Nessler methods are comparable to Conway method in concentrate samples, but are inconsistent in legumes, while the ISE method requires further validation for NH3 determination in rumen fermentation studies.