Time of rumen fluid collection relative to feeding alters in vitro fermentation gas parameters

The objective of this study was to determine the effect of time of rumen fluid collection relative to feeding on gas production kinetics for in vitro rumen fermentation. Three ruminally cannulated Holstein heifers were rumen fluid donors. Feed was removed from heifers 12 h prior to feeding, rumen fluid was collected from each heifer before feeding (0 h), and at 2, 4, and 6 h after feeding, repeated on three separate incubation days. Buffered rumen fluid (100 mL) was incubated in 250-mL bottles containing 1.4 g of dried TMR, in duplicate for each heifer at each collection time. All bottles were incubated for 24 h at 39°C and constant agitation (60 rpm), and capped with monitors to capture temperature and pressure every 15 min (RF1, Ankom Technology, Macedon, NY). At the end of incubation, final pH was recorded. Gas data were fit with nonlinear regression comparison of fit in GraphPad Prism 7 to find best fit regression. The formula with best fit was as follows: y = V_m(1 - (e(-K_d(x - lag)))), where y is gas produced at time X (mmol), V_m is the asymptotic gas production (mmol), K_d indicates the fractional rate of gas production (mmol/h), X is time (h), and lag refers to the lag time before the start of fermentation (h) as indicated by positive gas production (R ² = 0.98). Data were analyzed using PROC GLIMMIX of SAS, with donor as the experimental unit and day as the random blocking factor; significance is defined as P ≤ 0.05. Time of rumen fluid collection significantly affected gas production kinetics (lag P = 0.01, V_m P = 0.03, K_d P <0.0001). Gas production was highest in fermenter units fed with rumen fluid collected 2-h post-feeding. Fractional rate of fermentation (Kd) was fastest in rumen fluid collected at 0 h. Lag time was longest in rumen fluid collected at 4-h post-feeding and slowest in rumen fluid collected at 0 h. Time of rumen fluid collection did not alter final pH. Our findings suggest that gas production is maximized when rumen fluid is collected between 2 and 4 h after feeding.