An in vitro method based on ¹⁵N-labeled forage N was developed to study ruminal protein degradation of timothy and red clover. Timothy and red clover were grown on the same field with 2 replicate plots, 2 m² each. One replicate plot was fertilized with ¹⁵N-enriched NH₄NO₃ while the other received non-enriched fertilizer. Labeled timothy and red clover had average enrichment of 2.37 and 1.23 atom % excess ¹⁵N, respectively. Forages from the first-cut were preserved either as hay (TH, RCH), untreated (T, RC) or formic acid-treated silage (TF, RCF). Rumen fluid was collected from 2 Swedish red cows fed on grass silage:concentrate diet (60:40 DM basis). Samples of 1 g were incubated in 60 mL of buffered rumen fluid at 39°C for 48 h. For non-labeled forages NH₃-N in the inoculum was labeled with ¹⁵N enriched (NH₄)₂SO₄. Vessels containing labeled forages received the same amount of non-enriched (NH₄)₂SO₄. Liquid samples were collected every hour at 0 to 10 h, after 12, 16, 24, 30 and 48 h, and later analyzed for NH₃-N and ¹⁵N. Degraded CP was calculated as a sum of appearance of ¹⁵N from labeled forages and disappearance of ¹⁵N from labeled NH₃-N. Degradation parameters were estimated by the 2 pool exponential model using the SAS NLIN procedure. Timothy forages had higher rapidly degradable fraction (b1). Slowly degradable fraction (b2) was smaller in formic acid treated silages and in red clover silages. Hay had slower degradation rates (kd) than silages. Total degraded CP (a+b) was higher for timothy and for hay. It is concluded that incubating ¹⁵N labeled feeds or feed fractions simultaneously with corresponding unlabeled feeds by labeling ammonia pool with ¹⁵N can be a useful tool in investigating kinetics of ruminal protein degradation. Table 1. CP concentration and degradation parameters of timothy (T) and red clover (RC)

	TF	RCF	T	RC	TH	RCH
CP, g/kg DM	155	147	166	133	144	137
CP degradation1
b1, g/kg	495	418	335	334	473	391
b2, g/kg	380	362	594	507	490	452
kd1, 1/h	0.696	1.250	1.263	1.200	0.815	0.818
kd2, 1/h	0.189	0.132	0.139	0.161	0.055	0.091
a+b, g/kg	875	780	929	841	963	843

¹b1 = rapidly degradable, b2 = slowly degradable, kd1 = degradation rate of b1, kd2 = degradation rate of b2.