Date of Graduation

5-2013

Document Type

Thesis

Degree Name

Master of Science in Animal Science (MS)

Degree Level

Graduate

Department

Animal Science

Advisor/Mentor

Rosenkrans, Charles F. Jr.

Committee Member

Moubarak, Ali S.

Second Committee Member

Looper, Michael L.

Third Committee Member

Anthony, Nicholas B.

Keywords

Pure sciences; Biological sciences; Alkaloid; Bovine; Cytochrome; Ergot; Fescue; P450

Abstract

This thesis evaluates the PromegaTM P450-Glo assay (PromegaTM V9800) as a tool for quantifying ergot alkaloid concentration. Current techniques used for detection of ergot alkaloids are slow and expensive, do not detect all ergot alkaloids, or are not effective on bovine bodily fluids. The first study was conducted to determine effects of commercial ergot alkaloids (n = 6; 0 - 400 μM) on the PromegaTM P450-Glo assay. Cytochrome P450 (CYP450) activity in assay had a differential response to each ergot alkaloid and concentration. As concentrations of ergotamine, dihydroergotamine, ergocornine, and ergocryptine increased CYP450 activity was inhibited (P < 0.05). Increases in ergonovine and pergolide concentration did not affect (P > 0.1) CYP450 activity. These results verify that the PromegaTM P450-Glo assay is able to detect presence of ergot alkaloids. The second study was conducted to determine ability of the PromegaTM P450-Glo assay to detect ergot alkaloids in biological samples. Bovine urine and serum were analyzed with the PromegaTM P450-Glo assay to test for effects of forage and genotype. The single nucleotide polymorphism tested was designated by alu1 cleaving enzyme. Crossbred Angus-sired steers (n = 39; 216 ± 2.6 days; 203 ± 1.7 kg) were blocked by weight and assigned to graze toxic tall fescue (E+; n = 4) or non-toxic fescue (HM4; n = 4) pastures. After grazing 105 days animals were weighed, and blood and urine samples were collected. Samples were analyzed using the PromegaTM P450-Glo assay. Enzyme linked immunosorbent assay (ELISA) was used to test urine for total ergot alkaloids. Animals were genotyped for CYP450 single nucleotide polymorphism. Data were analyzed for correlations and one way analysis of variance. Genotype affected (P < 0.05) ability of urine to inhibit CYP450 activity, while forage did not. Genotype and forage did not affect ability of serum to inhibit CYP450 activity. Inhibition of CYP450 activity by urine was strongly correlated (r = -0.50; P < 0.025) with total ergot alkaloids in urine as determined by ELISA. Results indicate the PromegaTM P450-Glo assay is able to detect ergot alkaloid presence in urine.

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