Development and evaluation of new techniques to quantify ruminal pool size and duodenal flow of protozoal nitrogen
Currently used microbial markers cannot distinguish protozoal nitrogen (N) from bacterial N, so research on protozoal quantification in vivo has been limited by the lack of a repeatable, accurate marker for protozoal N. The first set of experiments was focused on the development of a real-time polymerase chain reaction assay targeting the gene encoding 18S ribosomal RNA (rDNA) to quantify the amount of protozoal biomass in ruminal fluid and duodenal digesta. The DNA from concentrated cells was extracted with virtually 100% efficiency both before and after column purification. After serial spiking of protozoal cells into duodenal fluid over the entire range of quantification, the recovery was highly linear and constant at 81%. The next experiments were designed to generate in vivo data as a preliminary test of the real-time PCR method’s robustness. The objectives were 1) to determine the degree of reduction of bacterial contamination using the new protozoal isolation techniques, 2) to determine if protozoal standards derived from ruminal fluid are appropriate for predicting duodenal flows, and 3) to evaluate the assay’s determined values for protozoal N in the rumen and flowing to the duodenum compared with independent measurements. The protozoal collection method reduced non-associated bacterial contamination by 33 fold. Protozoal N was determined to be 4.8 and 12.7% of the rumen microbial N pool and 5.9 and 11.9% of the duodenal flow of microbial N on diets containing low (16%) or high (21%) forage NDF, respectively. Final experiments were designed to determine if protozoal growth, rDNA, RNA and N are modulated by starvation or by the addition of the ionophore antibiotic monensin. In experiment 1, our objectives were to determine how abruptly changing transfer rate affects rumen protozoal growth with or without addition of monensin. In experiment 2, our objectives were to determine diurnal changes in protozoal rDNA, N, and RNA. With increased transfer rate, protozoal growth rate increased to avoid wash-out. Monensin treatment increased generation time, causing several species to die. Protozoa had differing copies of rDNA (P < 0.05) per cell that seem to depend on their cell size and to be regulated by nutrient availability.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:rumen protozoa 18s rdna real time pcr microbial nitrogen
Date of Publication:01/01/2005