Volatile basic nitrogen measurement in digesta using a Berthelot reaction in automated Skalar instrumentation

The undigested nitrogenous fraction entering the hindgut of chickens is further metabolized by microbiota present producing volatile basic metabolites including amines and ammonia (NH₃). Ammonia increases pH and may result in overgrowth of Clostridium perfringens further producing toxic metabolites that cause dysbacteriosis or necrotic enteritis (NE). There are few reports in chickens examining the production and concentration of nitrogenous metabolites in the hindgut. A Berthelot reaction using an automated flow, reaction and spectrophotometric instrumentation to detect volatile basic nitrogen in the form of NH₃ from cecal contents was carried out. In the Berthelot reaction, NH₃ in the sample is chlorinated to monochloramine using dichloroisocyanuric acid that then reacts with salicylate to form 5-aminosalicylate which is stable under alkaline conditions (pH 12 to 13). After oxidation and oxidative coupling, a coloured complex is formed that can be measured at 660 nm. Cecal contents were collected and pooled from 2 birds per pen with 48 pens total in each of 3 experiments and stored in sealed containers at -20 °C prior to analysis. Experiment 1 compared samples collected from birds fed either no meat and bone meal (MBM) or 6% MBM from d 0 to 14, and samples collected from birds fed either no MBM or 5% MBM from d 14 to 42. All birds were challenged with Eimeria on d 9 and C. perfringens on d 14 and 15. Experiment 2 compared cecal contents from birds fed either 0.5% or 0.9% calcium (Ca), and Exp. 3 compared unchallenged with NE challenge on d 16 and 29. Results demonstrated an increase (P < 0.05) in cecal NH₃ as a result of feeding MBM or high dietary Ca with a corresponding increase in pH. Birds experimentally challenged with NE had lower cecal NH₃ and lower pH on d 16 but not d 29 compared to unchallenged controls. It was concluded that the Berthelot method using Skalar instrumentation equipment is suitable to measure the concentration of volatile nitrogen as NH₃ in cecal contents of chickens.