phase could improve the subsequent viability of cells due to the protective effect of the cold shock that proteins produced. If so, a practical implication in the field of rumen inoculum preservation could be the investigation of optimal pre-cooling protocols prior to full refrigeration.

Refrigerated ru- men fluid could potentially re- duce the need for laboratories to maintain ani- mal donors and reduce the fre- quency of col- lecting rumen fluid.

Cooling rumen fluid for up to 96 hours Despite the similarity of fermentative metabolites with 96 hours of cooling, there was a decline in 24-hour gas produc- tion at 96 hours of refrigeration. However, these results were not unexpected based on Robinson et al. (1999) who found stable NDF degradability of fresh and 48-hour refrigerated (4°C) rumen fluid. Another study reported that chilling inocu- lum for 6 hours did not affect in vitro gas production, but 24 hours of refrigeration reduced the fermentation rate. In addi- tion, in two other studies the rumen fermentability of fresh rumen fluid was maintained, measured in terms of gas pro- duction, when it was preserved for approximately 4 hours at 4 to 6°C.

but there was a decrease (P<0.01) when rumen fluid was used after 96 hours of refrigeration. - Experiment 2 Cooling rumen fluid solids for 48 hours: The six rumen fluids had different physic-chemical characteristics (pH 6.1 + 0.4; ammonia 22.9 + 13.7 mg/dℓ L; total VFA 111.7 + 13.1 mmol/ℓ) and the pellet yield was 151 + 42 g/kg of the initial rumen fluid weight. There was an interaction between inoculum×feed substrate (P <0.01) for the rate of gas produc- tion, but the impact of the interaction to the mean square of the model was much lower than that of the corresponding main effects. Only the results of the main effects of the model are therefore shown and discussed. Maximum gas yield (asymp tote) was higher with fresh inoculum compared to that obtained from the pellet (286 vs 263 mℓ/g DM; P<0.01) whereas the rate of degradation showed the opposite pattern (0.072 and 0.093mℓ/h for fresh inoculum and pellet, respec- tively; P<0.01). In addition, the reconstituted liquid had a longer lag phase compared to fresh (0.70 vs 0.14 hours; P<0.01). Gas production at 24 hours was similar for fresh and pellet inoculum; in Figure 1 the linear regression between gas measured at 24 hours of fermentation from the fresh and pel- let inoculum (adjusted for the rumen fluid effect) is shown (R2 = 0.94).

Cold shock response The concentration of fermentation metabolites in rumen fluid was not changed by refrigeration, apart from an increase in ammonia concentration. Cold shock response is a complex re- action by which bacterial groups adapt to cold temperatures. This includes several metabolic processes, such as modifi- cation in membrane lipids and synthesis of proteins able to favour sugar metabolism. It is likely that the higher ammo- nia levels after refrigeration was due to increased protein metabolism due to cold. It is suggested that a refrigeration

40 ▶ ALL ABOUT FEED | Volume 29, No. 1, 2021

Cooling rumen fluid solids for 48 hours Centrifugation is widely used to separate rumen microbes from liquids and feed particulates in rumen fluid. Unfortu- nately, centrifugation protocols (e.g. centrifugal force, time, and temperature) are not standardised. However, in well- known studies researchers tested three centrifugation speeds (5, 10 and 26×103 x g and 5, 17 and 30×103 x g, re- spectively) for 20 or 30 minutes. As both groups reported no differences in the amount of ruminal bacteria separated by different centrifugations speeds, we used an intermediate value of 13×103×g. A previous study also used this centrifu- gation speed in preparing a rumen pellet to study the glyco- gen recovery from rumen microbes when comparing meth- ods for isolating rumen bacteria. In our study, the yield of wet pellet was variable (i.e. 151 + 42g/kg of the initial rumen fluid volume), which probably reflects differences in the par- ticulate density of the rumen fluid collected, despite using a common filtration procedure. Variations in pellet yield will be reflected in reconstituted rumen fluid, which requires the use of blanks (i.e. fermentation from bottles containing only fermentation fluid).

Conclusions Our results show that rumen fluid preserved by refrigeration (at 4°C until 72 hours) or reconstituted from a refrigerated bacterial pellet (at 4°C for 48 hours), does not depress 24- hour gas production. Refrigerated rumen fluid (or pellet) is an alternative to fresh fluid and would aid in the supply and transport of rumen fluid between laboratories, as well as the potential use of the same inoculum in repeated fermentation runs within the laboratory. This would reduce the need for laboratories to maintain animal donors and/or reduce the frequency of collecting rumen fluid.

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