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To reduce the propensity of alfalfa to cause pasture bloat in grazing animals, researchers sought to transform this popular forage with a bHLH anthocyanin regulatory gene (Lc) from maize. Expression of the Lc (leaf color) gene causes accumulation of anthocyanidin and proanthocyanidins in alfalfa tissues, giving rise to novel 'purple-green' forage. In this study, the effects of Lc expression on ruminal degradation of the forage were studied in vitro. Field-grown parental and three transgenic forages were harvested at the pre-bud stage, freeze-dried and ground (1.0 mm) for use as substrate. Anthocyanidin content in the transgenic genotypes ranged from 130 to 136 μg/g DM. The ground forages (500 mg in F57 bags) were incubated anaerobically for 0, 2, 4, 8, 12, 16, 24, and 48 h in a DAISYII Incubator (ANKOM Technology, Macedon, NY) for determination of DM and N degradabilities (n = 9). A second incubation was conducted (sampling at 0, 4, 8 and 12 h) with 15N included as a marker for microbial protein. Compared to the non-transgenic parental alfalfa (NT), the Lc-transgenic genotypes contained less (P = 0.034) rapidly soluble DM, but their slowly degradable DM fractions, rates of degradation and lag times for digestion were similar (P ≥ 0.122) to NT. At 0 h, the true disappearances of N and DM were lower (P < 0.001 and P = 0.027, respectively) from the Lc genotypes than from NT alfalfa, but they did not differ (P ≥ 0.137) at 4, 8, or 12 h. Solubility of DM and N were both negatively correlated (R2 ≥ 0.563) with the concentration of anthocyanidins measured in the forage. Lc-transformation reduced the initial rate, but not the extent of DM and N digestion of alfalfa in the rumen. These properties could improve the utilization of protein and possibly reduce the risk of bloat in ruminants consuming fresh alfalfa. [ABSTRACT FROM AUTHOR]