Samples were strained through 4 layers of cheese cloth, and pH was determined immediately with a portable pH meter (model miniLab IQ125; IQ Scientific Instruments, Inc., Carlsbad, CA); the fl uid portion was transferred into duplicate 50 mL plastic centrifuge tubes, and each tube was acidifi ed with 1 mL of 6 N HCl and immediately frozen (−20°C) until analyzed for VFA, Co, and rumen NH3