| Summary: | Storing rumen fluid (RF) has the potential to standardize subsequent in vitro feed fermentation studies. The first phase of this experiment aimed to evaluate the effect of two RF storage methods on gas composition and dry matter disappearance (DMD) in wheat grain and lucerne hay under in vitro fermentation. The storage methods were as follows: (1) snap-freezing RF using liquid nitrogen and then storing it at −80 °C (−80 °C); and (2) mixing RF with 5% dimethyl sulfoxide (DMSO), subsequently freezing it at −20 °C (D−20 °C), and comparing it to fresh RF on days 1, 14, and 30 post collection. The objective of the second phase was to quantify the impact of preserving the RF for 180 days at D−20 °C on the in vitro fermentation parameters. The methane composition was lower (<i>p</i> < 0.001) in both the preserved RFs than in the fresh RF. There was no difference (<i>p</i> < 0.05) in DMD values between days 14 and 30. The average cumulative gas production and DMD from the RF stored at D−20 °C was higher than that from the RF stored at −80 °C. Moreover, there was no difference between day 30 and day 180 in the total gas production and lag time when fermenting with RF preserved at D−20 °C. Therefore, storing RF at D−20 °C is preferable to storing it at −80 °C when access to fresh RF is limited.
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