The Effects of Animal, Collection Time, and Interval on the Microbiota Structure, Metabolism, and Degradative Potential of Rumen Fluid Inoculum Collected by Esophageal Probe from Hay-Fed Cows.

Rumen fluid (RF) is the greatest source of uncontrolled inter-assay variations in in vitro digestibility tests. This study aimed to investigate the effect of donor cow (DC) individual variability, time relative to feeding, and weekly sampling intervals on RF microbiota structure and metabolism, and on its degradative capacity expressed as enzymatic activity (EA). RF was collected using an esophageal probe from 4 Holstein hay-fed DCs before feeding, and 4 and 8 h post-feeding, over 3 days with one-week intervals, for a total of 3 weeks. Rumen bacteriome ß-diversity was affected by DCs and the one-week interval but not by time relative to feeding. However, volatile fatty acids and NDF and DM digestibility were not altered based on DCs. Propionic, lactic, and butyric acids were consistent between weeks, with some variations in lactic and butyric acids 8 h after feeding. Amylase and cellulase activities fluctuated between the weekly intervals, while xylanase activity increased in RF collected at 4 and 8 h after feeding ( = 0.030) and in DC3 compared to the other DCs ( < 0.001). Our results reinforce the importance of pooling RF from multiple cows and increasing the number of replicates to enhance the accuracy of the analysis.