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 nutrien t-gene interactions in dairy cows at the molecular level. A series of techniques was applied to study the molecular response of cells in bov ine small intestine to dietary manipulation. Three Holstein cows fitte d with ruminal and duodenal cannulae were used for the investigation. As part of the validation of the procedure, lipid flow to the small in testine was manipulated using an abomasal infusion technique to study the expression of the gene encoding for the gastrointestinal hormone c holecystokinin (CCK). Biopsies (10 to 25 mg) were collected from the d uodenum approximately 40 to 50 cm distal from the pyloric sphincter us ing a fiberoptic endoscope and biopsy forceps. Total RNA was isolated from the intestinal biopsies with yields of 20 to 50 mu g. Cholecystok inin gene expression was analyzed in intestinal mucosa obtained from H olstein cows after slaughter either at a local abattoir or at our rese arch facilities and compared with CCK gene expression in the biopsy sa mples collected from the cannulated animals. Using the technique of no rthern hybridization, we found that 15 mu g of total RNA was sufficien t to detect a strong CCK mRNA signal in the biopsy tissue samples, whe reas 30 mu g 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 detectabl e with 40 mu g of total RNA. We conclude that the biopsy procedure is relatively noninvasive and is useful for monitoring intestinal respons es to dietary manipulation and to physiological changes, such as those that occur during lactation and with metabolic disorders. The applica tion of the method will provide insight into nutrient-gene interaction s that occur in bovine intestinal mucosa.