Rumen fluid preservation for in vitro gas production systems

Preservation of rumen fluid is a potential approach to standardize in vitro studies while minimizing the cost of maintaining donor animals, variations in the rumen fluid collection procedure, and ethical issues. Using a batch gas production system, the present experiment evaluated a series of rumen fluid preservation techniques for their use in in-vitro feed fermentation. A factorial design was used to compare two substrates (lucerne hay vs. wheat grain) and four preservation techniques relative to fresh rumen fluid: (1) Frozen at − 20 ⁰C, (2) Preserved using 5 % dimethyl sulfoxide (DMSO) and frozen at − 20 ⁰C (D-20 ⁰C), 3) snap-frozen using liquid Nitrogen and stored at − 80 ⁰C (− 80 ⁰C), and 4) preserved using 5 % DMSO, snap frozen in liquid Nitrogen and stored at − 80 ⁰C (D-80 ⁰C), over five incubation periods (1, 4, 8, 14 and 30 days). The mean cumulative gas production of wheat grain (84.5 ml/g) and lucerne hay (54.9 ml/g) incubated with fresh rumen fluid did not differ (P > 0.05) from the gas production using rumen fluid preserved with D-20 ⁰C, 90.1 ml/g and 55.0 ml/g respectively. The maximum gas production from fresh rumen fluid (87.6 ml/g) was also not different (P > 0.05) from rumen fluid preserved with D-20 ⁰C (85.3 ml/g). The concentration of acetic acid, propionic acid, and isobutyric acid did not differ (P < 0.001) between fresh rumen fluid and the D-20 ⁰C. Incubated substrates produced higher average ammonia-N values (P > 0.05) when fermented with fresh rumen fluid compared to a preserved rumen fluid, except for − 80 ⁰C. The rumen fluid stored under D-20⁰C produced gas production results with the least difference to fresh rumen fluid and would be a more reliable preservation technique to adopt than the other methods evaluated.