Development of an in vivo method to study bovine intestinal response to dietary manipulation at the molecular level

Our objective was to develop an in vivo method for analysis of nutrient-gene interactions in dairy cows at the molecular level. A series of techniques was applied to study the molecular response of cells in bovine small intestine to dietary manipulation. Three Holstein cows fitted with ruminal and duodenal cannulae were used for the investigation. As part of the validation of the procedure, lipid flow to the small intestine was manipulated using an abomasal infusion technique to study the expression of the gene encoding for the gastrointestinal hormone cholecystokinin (CCK). Biopsies (10 to 25 mg) were collected from the duodenum approximately 40 to 50 cm distal from the pyloric sphincter using a fiberoptic endoscope and biopsy forceps. Total RNA was isolated from the intestinal biopsies with yields of 20 to 50 microg. Cholecystokinin gene expression was analyzed in intestinal mucosa obtained from Holstein cows after slaughter either at a local abattoir or at our research facilities and compared with CCK gene expression in the biopsy samples collected from the cannulated animals. Using the technique of northern hybridization, we found that 15 microg of total RNA was sufficient to detect a strong CCK mRNA signal in the biopsy tissue samples, whereas 30 microg of total RNA showed a slightly weaker CCK mRNA signal in the postmortem duodenal tissues collected at our research facility. In contrast, the CCK mRNA signal in postmortem duodenal tissues obtained from cows slaughtered at the local abattoir was only barely detectable with 40 microg of total RNA. We conclude that the biopsy procedure is relatively noninvasive and is useful for monitoring intestinal responses to dietary manipulation and to physiological changes, such as those that occur during lactation and with metabolic disorders. The application of the method will provide insight into nutrient-gene interactions that occur in bovine intestinal mucosa.