Abstract
The accuracy of two different methods in measuring the ammonia nitrogen (N-NH3) concentration in rumen fluid were evaluated: a catalyzed indophenol colorimetric reaction (CICR) and the Kjeldahl distillation (KD). Five buffered standard solutions containing volatile fatty acids, true protein, and known ammonia concentrations (0, 3, 6, 12, and 24 N-NH3 mg/dL) were used to simulate rumen fluid. Different ratios (10:1, 7.5:1, 5:1, 2.5:1, 1:1, 1:2.5, 1:5, 1:7.5, and 1:10) of a potassium hydroxide solution (KOH, 2 mol/L) to standard solutions were evaluated by the KD method. The accuracy of each method was evaluated by adjusting a simple linear regression model of the estimated N-NH3 concentrations on the N-NH3 concentrations in the standard solutions. When the KD method was used, N-NH3 was observed to be released from the deamination of true protein (P<0.05), and an incomplete recovery of N-NH3 was observed (P<0.05), except for 7.5:1 and 5:1 ratios of KOH solution to standard solutions (P>0.05). The estimates of the N-NH3 concentration obtained by the CICR method were found to be accurate (P>0.05). After the accuracy evaluation, ninety-three samples of rumen fluid were evaluated by the CICR and KD methods (using the 5:1 ratio of KOH solution to rumen fluid sample), assuming that the CICR estimates would be accurate. The N-NH3 concentrations obtained by the two methods were observed to be different (P<0.05) but strongly correlated (r = 0.9701). Thus, it was concluded that the estimates obtained by the Kjeldahl distillation using a 5:1 ratio of KOH solution to rumen fluid sample can be adjusted to avoid biases. Furthermore, a model to adjust the N-NH3 concentration is suggested.
Highlights
MATERIAL AND METHODSSeveral parameters have been suggested for evaluating the availability of dietary nitrogenous compounds in the rumen
Chaney and Marbach (1962) have proposed a method for N-NH3 evaluation in biological fluids that is based on a catalyzed indophenol colorimetric reaction (CICR), and this method is intensely used in microbiological assays (e.g., Thomas and Russell, 2004)
The quantity of the KOH solution used in the Kjeldahl distillation (KD) method must be sufficient to neutralize even extremely high concentrations of volatile fatty acids (VFA); yet, the solution should remain weak enough to prevent the deamination of major amino acids (Fenner, 1965)
Summary
MATERIAL AND METHODSSeveral parameters have been suggested for evaluating the availability of dietary nitrogenous compounds in the rumen. The concentration of the ammonia nitrogen (N-NH3) has been used as a qualitative reference to understand the adequacy of the rumen environment according to the microbial activity on fibrous carbohydrates (Detmann et al, 2009). Fenner (1965) has established the theoretical basis for N-NH3 evaluation in rumen fluid by steam distillation in the presence of a potassium hydroxide solution This basis has been adapted for use in the Kjeldahl distillation (KD) procedure, which has been used in several ruminant nutrition assays (e.g., Detmann et al, 2009; Souza et al, 2010; Costa et al, 2011). No study has compared the accuracy of these methods in the estimation of N-NH3 contents
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