Development and validation of a real-time PCR method to quantify rumen protozoa and examination of variability between entodinium populations in sheep offered a hay-based diet

Abstract

PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR (epsilon) was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R2= 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (10(0) and 10(6) entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P = 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required.

FIG. 1.

Example of real-time PCR amplification of serially diluted rumen DNA to calculate ɛ from the slope of the linear-regression plot (threshold cycle versus log dilution) according to the equation ɛ = 10^−1/slope. In this example, the slope is −3.82, and the PCR efficiency is 1.82.

FIG. 2.

Correlation between Entodinium populations in the sheep rumen measured by microscopic counts and real-time PCR. The data are presented as log numbers of Entodinium cells per gram (wet weight) rumen contents. The real-time PCR primers used were Oph-151F and Ento-472R. Standard errors ranged from 0.8 to 11%.