Evaluation of a Real-Time PCR Assay Quantifying the Ruminal Pool Size and Duodenal Flow of Protozoal Nitrogen

We have recently developed a real-time polymerase chain reaction (PCR) assay to quantify copies of the genes encoding protozoal 18S rRNA. The assay includes procedures for isolating and concentrating protozoal cells from the rumen for use as a standard to convert 18S rRNA gene copies to a biomass ba...

Full description

Saved in:
Bibliographic Details
Published in:Journal of dairy science 2005-06, Vol.88 (6), p.2083-2095
Main Authors: Sylvester, J. T, Karnati, S. K. R, Yu, Z, Newbold, C. J, Firkins, J. L
Format: Article
Language:English
Subjects:
Animal productions
Animals
Biological and medical sciences
Cattle
dairy cows
denaturing gradient gel electrophoresis
Diet
Dietary Fiber - administration & dosage
digestible protein
Digestion
Duodenum - metabolism
Eukaryota - isolation & purification
Eukaryota - metabolism
Female
filtration
Food industries
Fundamental and applied biological sciences. Psychology
microbial detection
Milk and cheese industries. Ice creams
Nitrogen - analysis
Nitrogen - metabolism
Polymerase Chain Reaction - methods
real-time PCR
ribosomal RNA
RNA, Ribosomal, 18S - genetics
rRNA
Rumen - chemistry
Rumen - metabolism
Rumen - parasitology
rumen protozoa
rumen protozoal nitrogen
Terrestrial animal productions
Vertebrates
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:We have recently developed a real-time polymerase chain reaction (PCR) assay to quantify copies of the genes encoding protozoal 18S rRNA. The assay includes procedures for isolating and concentrating protozoal cells from the rumen for use as a standard to convert 18S rRNA gene copies to a biomass basis. The current objectives were to 1) determine the degree of reduction of bacterial contamination in the protozoal standard, 2) determine if protozoal standards derived from ruminal fluid are appropriate for predicting duodenal flows, and 3) evaluate the assay's determined values for protozoal N in the rumen and flowing to the duodenum compared with independent measurements. Our protozoal collection method reduced non-associated bacterial contamination by 33-fold, the contamination of which could otherwise significantly bias RNA (microbial marker) and N percentages of concentrated protozoal fractions. Based on denaturing gradient gel electrophoresis, the use of protozoal cells isolated from ruminal fluid appears appropriate for use in quantitative assays determining protozoal N flow postruminally. Using real-time PCR, 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 neutral detergent fiber, respectively, which were comparable with independent measures and expectations.
ISSN:0022-0302
1525-3198
DOI:10.3168/jds.S0022-0302(05)72885-X