Effects of preservation procedures of rumen inoculum on in vitro microbial diversity and fermentation

Sheep rumen contents were used as inoculum for an in vitro semi-continuous incubation system to study whether preservation method affects microbial fermentation pattern. Rumen fluid was filtered and either used immediately as inoculum (CTL) or dispensed into 110 mm × 16 mm tubes, that were stored refrigerated at 6 °C for 4 h (REF) or frozen at −20 °C (FRZ), frozen in liquid N (FLN) or added with 0.04 glycerol and frozen in liquid N (FGL) for 48 h. Frozen inocula were thawed at 39 °C for 2 min before use (16 ml per bottle). Two 24 h incubations with four bottles per treatment were completed. The microbial utilisation of added glycerol after thawing in FGL increased total gas production (P<0.05) and 24 h volatile fatty acid (VFA) production (P<0.05), and also increased propionate and butyrate proportions at the expense of acetate. The other freezing inocula (i.e., FLN and FRZ) reduced the rate of gas production (as ml/g dry matter per hour), compared with CTL in the first 2 and 4 h of incubation (P<0.05), but this was compensated by increased fermentation at 8 and 12 h, respectively. Differences in gas production did not manifest a different VFA pattern at either 6 or 24 h incubation. Bacterial diversity was slightly affected by the preservation process, and the similarity index between untreated inocula and the 24 h incubated CTL samples was 0.690–0.724. Similarity between bacterial communities in FRZ and FLN with that in CTL after incubation was 0.678. The freezing preservation method of rumen inocula for subsequent in vitro gas production studies does not affect microbial fermentation pattern or bacterial biodiversity, provided that processing is rapid enough by using a high surface to volume ratio. Freezing in liquid N is more appropriate than at −20 °C.