Differential gene expression of ewes varying in tolerance to dietary nitrate

R. R. Cockrum, K. J. Austin, J. W. Kim, J. R. Garbe, S. C. Fahrenkrug, J. F. Taylor, K. M. Cammack

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Ruminants consuming diets with increased concentrations of nitrate (NO3-) can accumulate nitrite (NO2-) in the blood, resulting in toxicity. In a previous experiment, ewes identified as highly tolerant to subacute dietary NO3- were able to consume greater amounts of NO3- than lowly tolerant ewes without exhibiting signs of toxicity. We hypothesized that highly tolerant and lowly tolerant ewes differ in their ability to metabolize NO3- and thereby differ in the expression of hepatic genes involved in NO3- 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 quantity of dietary NO3-. Analysis of the Bovine Oligonucleotide Microarray data identified 100 oligonucleotides as differentially expressed (P < 0.05) between lowly tolerant 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 reversetranscription PCR. Relative expression, genomic regulation, and FC agreed between microarray and real-time reverse-transcription-PCR analyses, and FC differences (P < 0.05) between lowly tolerant and highly tolerant 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 dehydrogenase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase1, 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 haptoglobin, 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 NO3-.

Original languageEnglish (US)
Pages (from-to)3187-3197
Number of pages11
JournalJournal of animal science
Volume88
Issue number10
DOIs
StatePublished - Oct 2010

Keywords

  • Gene expression
  • Nitrate
  • Sheep
  • Toxicity

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