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An  in vitro  method based on  15 N-labelled forage nitrogen (N) was developed to study ruminal N metabolism of soluble N (SN), insoluble N (ISN) and neutral detergent insoluble N (NDIN) fractions of timothy forage. Timothy grass was grown on replicated experimental plots with one plot receiving  15 N-labelled and the other unlabelled N fertilizer. Harvested grass was preserved as dried grass or as formic acid treated or untreated silage. The intact forages and their corresponding N fractions were incubated in buffered rumen fluid  in vitro  to determine degradation parameters based on the  15 N fluxes between labelled feed N and ammonia N pools. A high percentage (25–38%) of  15 N-labelled ammonia disappeared from ammonia N pool during the first 15 min of incubation. Microbial uptake of dried grass SN fraction was higher than of silage SN fractions. Fractional degradation rates of SN from formic acid treated silage, untreated silage and dried grass during the first 6 hours of incubation were 0.145, 0.125 and 0.115 /h, respectively. By the end of the incubation period (28 h), 69, 66 and 43%, of the SN fraction of formic acid treated silage, untreated silage and dried grass, respectively were recovered as ammonia. The percentage of ISN fractions degraded to ammonia N were 9, 34 and 27%, respectively. Based on the changes in  15 N-labelled ammonia N pool in blank incubation and appearance of  15 N to ammonia N pool from  15 N-labelled NDIN fractions, it was estimated that a significant portion of microbial lysis occurred when incubations were carried out for longer than 20 hours. With dried grass the contribution of ammonia N for microbial N synthesis was greater than with silages. Use of  15 N-labelled forages together with this  in vitro  method is a promising technique for determining soluble N degradation parameters, but it requires further development to be used for determining degradation parameters of insoluble N fractions and work with whole feeds.

In vitro investigation of the ruminal digestion kinetics of different nitrogen fractions of 15N-labelled timothy forage