To test if branched-chain VFA (BCVFA) supplements can be reduced to as few as one BCVFA, combinations of BCVFA + valerate were replicated with different substrates in batch cultures of mixed rumen microbes. In Experiment 1, treatments consisted of 1) control, 2) isovalerate (Ival) + isobutyrate (Ibut) + 2-methylbutyrate (2MB) + valerate (Val; 1 mM each), 3) Ival + Ibut + 2MB (1.33 mM each), 4) Ival + Ibut (2 mM each), 5) Ival + 2MB (2 mM each), 6) Ibut + 2MB (2 mM each), 7) Ival (4 mM), 8) Ibut (4 mM), or 9) 2MB (4 mM). In 4 runs with 3 replicates, 30 mL of a 1:4 dilution of rumen fluid (blended to enrich particle-associated bacteria) in artificial saliva were anaerobically added to 50-mL tubes with either: 1) 0.5 g of alfalfa hay, 2) 0.25 g of alfalfa hay + 0.25 g of ground corn, 3) 0.5 g of grass hay, or 4) 0.25 g of grass hay + 0.25 g of ground corn in a 2 × 2 factorial arrangement of treatments. Model had the fixed effect of BCVFA dose, substrate, their interaction and the random effect of run. There was no feed by treatment interaction for NDF degradation (P = 0.31). All treatments increased (P < 0.01) NDF degradation compared with control, with no difference between any of the acid treatments (P > 0.10). Total VFA net production was not affected by treatment. In Experiment 2, treatments were the same, but 1mM of each acid was added (total BCVFA concentration varied among treatments) to study if compensation of dose affected results of Experiment 1. Grass + corn or alfalfa + corn were the sole substrate sources. There was a feed by treatment interaction for NDF degradation. When alfalfa + corn was the substrate, adding BCVFA did not improve NDF degradation (P = 0.14). When grass + corn was the substrate, adding 2MB alone, the combination of Ibut + Ival + 2MB + Val, Ibut + 2MB, or Ival + Ibut increased (P < 0.05) NDF degradation. Adding valerate was not beneficial. Number of BCVFA supplemented could be reduced to any combination if 4 mM of total acid was added, and the amount supplemented could be reduced when grass was the substrate; however, BCVFA were not as effective when alfalfa was the substrate, likely because of its higher protein content.