Biohydrogenation (BH) of fatty acids (FA) from fresh alfalfa and alfalfa hay with varying levels of supplemental sucrose and media pH was evaluated in vitro. A multicompartmental model was then developed to estimate pool size and flux of vaccenic acid (VA) during BH of FA in fresh alfalfa. To vary incubation pH, alfalfa samples were inoculated with rumen fluid in 2 media differing in molarity of the bicarbonate buffer. Samples were incubated for 0, 1, 2, 3, 4, 6, 9, and 12 h; pH was measured and tubes were put in ice and stored until analysis. The BH rates of linoleic acid (18:2) and linolenic acid (18:3) were estimated by PROC NLIN of SAS (single pool, first-order kinetic model) and SAAM II (multiple pools, first-order kinetic model). Both methods gave similar estimates for the BH rates of 18:2 and 18:3 as well as the temporal pool size of VA. The BH rates (/h) in the strong (SB) and weak buffers (WB) were 27.4 (± 0.7) and 23.5 (± 0.9) for 18:2, and 43.8 (± 0.2) and 30.3 (± 0.6) for 18:3, respectively. The WB decreased the BH rates of 18:2 and 18:3 for both forage sources. However, BH rates of 18:3 were higher from fresh alfalfa than alfalfa hay. There was no effect of sucrose addition on the BH rates of 18:2 and 18:3. Moreover, there was no effect of buffer on the BH of VA estimated by the multiple pools model between the SB and WB (12.5 ± 2.1 and 14.1 ± 3.7/h, respectively). The BH rates of the conjugated linoleic acid isomers were not different between the SB and WB treatments (36.7 ± 19.8 and 25.9 ± 27.2, respectively). Because we could estimate fluxes as well as mass of the VA pools, more information was generated from the data when a multiple pools model was used compared with a single pool, first-order kinetic model.
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