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This study investigated successional colonization of perennial ryegrass (PRG) by the rumen microbiota. PRG grown for 6 weeks in a greenhouse was incubated in sacco in the rumens of three Holstein × Freisian cows over a period of 24 h. PRG incubated within the rumen was subsequently harvested at various time intervals postincubation to assess colonization over time. DGGE-based dendograms revealed the presence of distinct primary (0–2 h) and secondary (4 h onwards) attached bacterial communities. Moving window analysis, band number and Shannon–Wiener diversity indices suggest that after 2 h a proportion of primary colonizing bacteria detach, to be replaced with a population of secondary colonizing bacteria between 2 and 4 h after entry of PRG into the rumen. Sequencing and classification of bands lost and gained between 2 and 4 h showed that the genus Prevotella spp. was potentially more prevalent following 4 h of incubation, and Prevotella spp. 16S rDNA-based QPCR supported this finding somewhat, as 2- to 4-h Prevotella QPCR data were greater but not significantly so. Low-temperature scanning electron microscopy showed that attached bacteria were predominantly enveloped in extracellular polymeric substances. In conclusion, colonization of fresh PRG is biphasic with primary colonization completed within 2 h and secondary colonization commencing after 4 h of attachment in this study.We investigated, over a 24-h period in sacco, whether attachment of rumen microbiota to perennial ryegrass (PRG) showed successional changes in diversity. Knowledge of the bacterial species that attach to PRG over time may aid our understanding of the temporal function of the attached microbiota and ultimately permit the development of novel strategies for improving animal production to meet the future demands for meat and milk.