Ruminant Nutrition: Dairy: Ruminal Fermentation and Health

Treponema saccharophilum was isolated in the early decades and described as highly preferred to utilize pectin for growth. Typically, alfalfa hay contains 10–15% pectin as a component of the cell wall matrix, whereas grass or corn stover has pectin content less than 5%. The objective of this study was to develop a set of PCR primers targeting the 16S rRNA gene of T. saccharophilum and to assess the relative abundance of T. saccharophilum associated with different forages that vary in pectin content using real-time PCR method. Fifteen 16S rRNA gene sequences having higher than 97% similarity with T. sacchrophilum, retrieved from our pyrosequencing data, were used as target templates along with type strain T. saccharophilum DSM2985 for primer designing. To confirm the specificity of our designed primers, 23 clones of the PCR amplicons amplifying from total rumen DNA were randomly picked and sequenced to build phylogenetic tree. Phylogenetic analysis revealed 2 closely-related clusters: 3 clones grouping with the type strain and the rest of clones grouping together with no cultivable representatives, suggesting the existence of a subspecies of T. saccharophilum. Growth of T. saccharophilum under in vitro system was compared among alfalfa hay (AH), Chinese wild rye hay (CW) or corn stover (CS) alone and in combination with pectin or corn starch as substrate, to investigate the efficiency of this method. After incubation for 24 h, T. saccharophilum grew better in AH or CW with pectin than CW or CS as sole fiber source (P < 0.05). Supplementation of corn starch did not enhance the growth of T. saccharophilum. The validation of this method was also investigated in vivo. Samples of rumen fluid were collected from 12 primiparous Chinese Holstein cows that were fed on diets with AH, CW or CS as main forage source. The results of real time-PCR showed that the relative abundance of T. saccharophilum in rumen of cows fed on AH as forage source was significantly higher than those on CW and CS (P < 0.05).