2010-Cockrum_et_al - Differential gene expression of ewes...

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M. Cammack R. R. Cockrum, K. J. Austin, J. W. Kim, J. R. Garbe, S. C. Fahrenkrug, J. F. Taylor and K. Differential gene expression of ewes varying in tolerance to dietary nitrate doi: 10.2527/jas.2009-2709 originally published online June 18, 2010 2010, 88:3187-3197. J ANIM SCI http://jas.fass.org/content/88/10/3187 the World Wide Web at: The online version of this article, along with updated information and services, is located on www.asas.org by guest on October 10, 2011 jas.fass.org Downloaded from
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ABSTRACT: Ruminants consuming diets with in- creased concentrations of nitrate (NO 3 ) can accumu- late nitrite (NO 2 ) in the blood, resulting in toxicity. In a previous experiment, ewes identified as highly tolerant to subacute dietary NO 3 were able to consume greater amounts of NO 3 than lowly tolerant ewes without ex- hibiting signs of toxicity. We hypothesized that highly tolerant and lowly tolerant ewes differ in their ability to metabolize NO 3 and thereby differ in the expression of hepatic genes involved in NO 3 metabolism. Therefore, our objective was to identify hepatic genes differentially expressed between ewes classified as lowly tolerant and highly tolerant after administration of a subacute quan- tity of dietary NO 3 . Analysis of the Bovine Oligonucle- otide Microarray data identified 100 oligonucleotides as differentially expressed ( P < 0.05) between lowly toler- ant and highly tolerant ewes. Functional analysis of the genes associated with these oligonucleotides revealed 2 response clusters of interest: metabolic and stress. Genes of interest within these 2 clusters (n = 17) and nonclustered genes with the greatest fold changes (FC; n = 5) were selected for validation by real-time reverse- transcription PCR. Relative expression, genomic regu- lation, and FC agreed between microarray and real-time reverse-transcription-PCR analyses, and FC differences ( P < 0.05) between lowly tolerant and highly toler- ant ewes were confirmed for 12 genes. Metabolic genes that were downregulated ( P ≤ 0.032) in lowly tolerant ewes vs. highly tolerant ewes included aldehyde oxidase 1, argininosuccinate lyase, putative steroid dehydro- genase, 6-phosphofructo-2-kinase/fructose-2,6-biphos- phatase1, and sterol carrier protein 2. In contrast, the metabolic gene homeobox was upregulated ( P = 0.037) in lowly tolerant ewes. The glutathione peroxidase 3 and inter-α (globulin) inhibitor H4 genes in the stress response cluster were upregulated ( P ≤ 0.045) in lowly tolerant ewes. Genes with the greatest FC, but did not cluster within the functional analysis included hapto- globin, which was upregulated ( P = 0.024) in lowly tolerant ewes, and fatty acid desaturase 2 and thyroid hormone responsive, both of which were downregulated ( P ≤ 0.019) in lowly tolerant ewes. Results from this study indicate that hepatic gene expression differs in ewes identified as lowly tolerant and highly tolerant to increased dietary NO 3 . Key words:
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2010-Cockrum_et_al - Differential gene expression of ewes...

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