Validation of sheep rumen fluid frozen in liquid N as inoculum for in vitro gas production trials.
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The effect of freezing rumen fluid in liquid nitrogen for its use as inoculum for in vitro incubation was evaluated in a semi-continuous system. Inoculum was used immediately (RF) or frozen and then thawed and used (TW) or preincubated for 2 (TW2) or 4 h (TW4) before substrate addition. For TW4, a sugars mixture was added. Bacterial diversity was not greatly affected by the inoculum processes. There were no differences on cumulative gas production (ml/g DM) or rate of gas production (ml/g DM per h) between RF and TW along the 36 h incubation (P>0.05). Delayed inocula (TW2 and TW4) produced less gas than RF up to 12h (P<0.05), but differences in fermentation rate were only appreciable at 2, 20 and 24 h (P<0.05). Volatile fatty acids (VFA) concentration at 6 h was higher with RF than TW and TW4, being lowest with TW2 (P=0.007) whereas at 24h it tended (P=0.07) to be higher with TW than with TW2 and TW4. Butyrate and minor VFA proportions differed for RF and frozen treatments at 6 and 24h (P<0.05). Despite of this, rumen fluid frozen in liquid N behaved similarly than fresh inoculum in terms of in vitro substrate fermentation. The lower gas volume in TW2 and TW4 might be justified by soluble nutrients utilisation during the first 2 and 4h incubation.