Rumen Metabolism of 22:6n‐3 In Vitro is Dependent on its Concentration and Inoculum Size, but Less Dependent on Substrate Carbohydrate Composition

Ruminal disappearance of linoleic and linolenic acid has been studied extensively. Less is known of the metabolism of docosahexaenoic acid (22:6n-3). The aim of this study was to identify factors which affect the disappearance of 22:6n-3 during in vitro batch incubations using rumen fluid from sheep. In experiment 1, the effect of the rumen fluid/buffer ratio (0.2 or 0.4), substrate (cellulose or cellulose/glucose), time of 22:6n-3 addition (0.08 mg/mL after 0 or 6 h of incubation) and incubation time (24 or 48 h) was evaluated. A mixture design was used in experiment 2 to evaluate the effect of carbohydrate type (cellulose, glucose, cellobiose and starch) on 22:6n-3 disappearance (0.08 mg/mL). In experiment 3, several concentrations of 22:6n-3 (0.05-0.30 mg/mL) were evaluated with different substrate mixtures (combinations of cellobiose, starch and cellulose). In a final experiment, the effect of the rumen fluid/buffer ratio (0.20, 0.35 and 0.50) and substrate (glucose, cellobiose and starch) was evaluated. In this experiment, 22:6n-3 was added as a proportion of rumen fluid ranging from 0.1 to 0.4 mg/mL rumen fluid, contrary to former experiments where concentrations were relative to culture medium. Low levels of 22:6n-3 (0.05 mg/mL) allowed extensive metabolism whereas increasing amounts of 22:6n-3 hampered its disappearance. A greater proportion of rumen fluid resulted in increased disappearance of 22:6n-3. The effect of carbohydrate type was small compared with the former two factors. These results suggest that in vitro metabolism of 22:6n-3 is mostly dictated by the conditions at the start of the incubation, i.e., inoculum, probably reflecting the density of bacteria able to metabolize 22:6n-3.