Determining Potential for on-Farm Fecal Collection for DNA Extraction

Determining the diet composition of cattle at pasture has proven to be extremely difficult and very time consuming to perform with accuracy. Some procedures are showing promise, yet with cover crop pastures with known plant species, a lower cost alternative might be use of qRT-PCR. The purpose of this project is to determine DNA concentration differences based on fecal collection processes to be used for further laboratory procedures. Four Holsteins, 3 beef heifer calves, and 4 pregnant beef cows were fed a controlled diet of 20% alfalfa, 50% fescue, 20% corn, and 10% soybean meal on a DM basis at 1.68% of body weight for 21-d. Between d 22 and 24 in the morning (8 AM) and afternoon (3 PM), and the AM on d 25, samples were taken FRESH from cattle or the pen for a DRY sample. DNA was extracted, purity and concentration were assessed using micro volume plate reader. Out of 105 isolated DNA samples, 89 samples proceeded to a secondary clean-up step and purity ratio 260/280 increased >1.4. The purity ratio less than 1.5 indicate potential protein contamination. Fecal DNA quality was not impacted by freshness of sample, time of day of collection, nor animal classification (P > 0.10). There was a tendency (P < 0.10) for fecal DNA quality to be purer in FRESH samples collected from cows versus DRY samples from Holsteins. Additionally, fecal DNA quality tended (P < 0.10) to be purer for FRESH samples taken in the PM versus DRY samples collected in the AM. In regards to extracted DNA concentration, there was no difference observed (P > 0.10) if samples were collected FRESH or DRY; in the AM or PM; nor differences based on cattle classification. However, FRESH samples taken in the PM yielded more DNA (P < 0.05) than DRY samples collected in the AM. There was also a tendency (P < 0.10) that a FRESH sample taken from a cow would generate more DNA than a DRY sample collected from Holsteins. All this taken into account, there are minimal differences in sampling methods based on animal classification, freshness of the sample, and time of day collection. Even so there is evidence that in a greater number of samples a FRESH sample collected in the afternoon would net the highest quality and greatest yields of DNA to be used for downstream use in qRT-PCR.