|Abstract:||Three studies with five ruminal-, duodenal- and ileal-cannulated sheep in a 5 x 5 Latin square design were conducted to test the possibility of manipulating nutrient flow and digestion of defaunated sheep. The first study evaluated effects of urea (U) and sodium bicarbonate (B) supplementation. Control sheep were faunated (treatment 1), with soybean meal (SBM) as the major N supplement. Remaining sheep were defaunated and fed either the same diet as treatment 1 (treatment 2), with U replacing 30% of the SBM-N (treatment 3) or with 2% B in the diet (treatment 4). Treatment 5 was a combination of treatments 3 and 4. Compared to the faunated control, defaunation decreased total tract organic matter (OM), fiber and crude protein (CP) digestibilities, but increased flow to and digestion in the small intestine (SI) of linoleic and linolenic acids. Urea supplementation improved total tract OM digestibility and B supplementation increased total tract fiber digestibility in defaunated sheep. The second study evaluated the applicability of using a combination of defaunation with N supplements (SBM, corn gluten meal (CGM), blood meal (BM), U and casein) of varying ruminal degradabilities to manipulate ruminal microbial protein synthesis and amount of ruminal escape protein. Treatment 1 (control) involved feeding faunated sheep a diet where the supplemental N was 2/3 SBM-N and 1/3 U-N. Treatments 2, 3, 4 and 5 involved feeding defaunated sheep diets where the supplemental N source was either 2/3 SBM-N:1/3 U-N, 2/3 CGM-BM-N:1/3 U-N, 1/3 CGM-BM-N:2/3 U-N or 1/3 CGM-BM-N:1/3 U-N:1/3 casein-N, respectively. Compared to the control, defaunation (treatment 2, 3, 4, and 5) decreased ruminal ammonia concentration and increased bacterial and non-bacterial CP flow to the duodenum and apparent amino acid (AA) digestion in the SI with no negative response to high U level (treatment 4) or positive response to casein supplementation (treatment 5). The third study was an extension of the second study. Compared to the control, all defaunation treatments increased ruminal bacterial density and protease activity against soluble protein, but had no consistent effect on deaminase activity. Defaunation also resulted in lower total perchloric acid-soluble amino-N with similar total free AA concentrations in ruminal fluid.