laboratory within 30 minutes from slaughter in airtight glass bottles refluxed with carbon dioxide and maintained at 39°C. - In experiment 1, a total of approximately 5ℓ of rumen fluid was collected from four cows, mixed and then divided into five equal aliquots. The first fraction was used immedi- ately for gas production, while the other four fractions were stored in a refrigerator at 4°C in closed bottles flushed with CO2. The aliquots were stored for 24, 48, 72 and 96 hours and then warmed at 39°C, sampled for pH, ammonia and volatile fatty acid (VFA) analysis, and used as inoculum for gas production fermentations. The experiment was repeated three times. - In experiment 2, rumen fluids were collected from three cows (1ℓ per cow) and divided into two equal aliquots. The first aliquot from each cow was used immediately for gas pro- duction and the others were weighted and centrifuged at 13,000×g for 30 minutes at 4°C. The supernatant was dis- charged, the residual pellet weighed to calculate pellet yield and then refrigerated at 4°C. After 48 hours the three pellets were reconstituted to a volume equal to that of the original rumen fluids using warm (39°C) McDougall’s buffer, with 5g/ℓ of added maltose, and were used as fermentation inoculum. The experiment was repeated twice. In both experiments, the in vitro evaluations were completed on maize grain meal, soya bean meal, wheat grain meal, de- hydrated lucerne meal, and a total mixed ration (TMR). Sam- ples of the TMR were dried at 60°C for 48 hours and all feed samples were milled through a 1mm sieve. Approximately


220mg of each dried sample was weighed into three graduat- ed 100mℓ glass syringes, which were filled with 30mℓ of di- lute rumen fluid. Syringes were placed vertically in a water bath at 39°C, with three syringes without substrate as blanks.


Measuring gas production Gas production was measured at 24 hours in experiment 1 and at 0, 1, 2, 4, 6, 8, 24 and 48 hours after incubation in ex- periment 2. Syringes were manually agitated every 2 to 8 hours of incubation, and then at 24 hours. In both experi- ments, we compared the gas production of different aliquots of the same rumen fluid in subsequent incubation sessions (e.g. fresh, refrigerated at 24 hours, refrigerated at 48 hours, etc.). To account for the incubation session effect, standard rumen fluid was included in each incubation session within each fermentation run.


Results of the study The fermentation of the standard rumen fluid gave similar gas yields among incubation sessions in both experiments (variations less than 5% not shown) and the data was therefore not corrected. - Experiment 1 Cooling rumen fluid for up to 96 hours: Refrig- eration did not modify total VFA yield, VFA proportions and pH, but increased (P<0.05) the ammonia level in rumen fluids refrigerated for 48, 72 and 96 hours compared to fresh (36,6 to 44,7 vs 23.4mg/dℓ). Gas yields from rumen fluid refrigerated for 72 hours did not differ from fresh inoculum,


▶ ALL ABOUT FEED | Volume 29, No. 1, 2021 39


Rumen fluid is used as a fresh inoculum for in vitro fermentations to predict the nutritional value of feed and rations for ruminants.


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