Unprotected long-chain polyunsaturated fatty acids, such as the n-3 fatty acids DHA and EPA are considered unsuitable for inclusion in ruminant feeding programs due to biohydrogenation by rumen microbes resulting in milk fat depression and inefficient transfer to milk. The aim of the current study was to investigate the effects of feeding an unextracted DHA-rich microalgae on the rumen microbiota and on feed digestibility in an in vitro batch culture rumen fermentation model. Five treatment levels (0, 2.5, 6.25, 15, or 25 mg) of a heterotrophically grown Aurantiochytrium limacinum (CCAP 4087/2, ALL-G-RICH, Alltech Inc.) biomass were added to vessels containing 0.25 g of 60:40 grass silage: commercial compound feed mix. Each treatment was replicated 15 times, with 5 replicate fermentation vessels of each treatment analyzed at 3 different time points (0, 4 and 8 h). Simulation vessels were flushed with CO₂ after which 9 mL of anaerobic buffer solution (38°C) was introduced under oxygen-free CO₂ flow. The simulation was initiated following the addition of 1 mL of freshly strained rumen fluid from a rumen fistulated dairy cow. Fermentation kinetics were followed by withdrawing vessels for quantifying volatile fatty acids (VFA), lactic acid and DHA after 0, 4 and 8 h post-inoculation. Gas production was measured as an indicator of microbial metabolic activity. After 8 h fermentation, bacterial yield was determined by quantitative RT-PCR. Each treatment was compared with the Control (no algae) group using 2-tailed t-tests. The addition of algae had no effect on gas, VFA, or lactic acid production and had no effect on the growth of bacteria. DHA was reduced in proportion to the starting concentration, with reductions of 21, 14, 10, and 1% observed for the 2.5-, 6.25-, 15-, and 25-mg treatments respectively. The unextracted algal biomass appeared to protect the DHA from biohydrogenation by ruminal microbes. As the biohydrogenation capacity is higher in vivo, these results warrant further investigation in live animals.