Lactating dairy cows convert 25 to 35% of intake N to milk N, and a part of the remaining N ends up in the environment, causing pollution. Dairy cows absorb amino acids available in the small intestine supplied mainly by digestion of microbial protein and ruminally undegraded feed protein (RUP). Ruminally degradable feed protein (RDP) is the major supplier of N for microbial protein synthesis. Most of the excess RDP will be degraded to ammonia and eliminated as urea in urine. Thus, avoiding excess RDP in dairy cattle diets is important in reducing environmental N pollution. The objectives of the work in this dissertation were to test the hypothesis that lactating dairy cows, when fed varying dietary RDP, can maintain feed intake, milk and milk protein yield, ruminal metabolism, passage of nutrients out of the rumen, and N excretion. The first study investigated the effects of decreasing RDP in lactating dairy cow diets on feed intake, milk production and apparent N efficiency. Forty mid-lactation cows (36 Holstein and 4 Jersey × Holstein cross-breds) were fed a diet containing 11.3% of diet dry matter (DM) as RDP for the first 28 d (covariate period). From d 29 to 47 (treatment period) cows were randomly assigned to 1 of 4 diets containing constant RUP (7.1% of DM) but 11.3, 10.1, 8.8, or 7.6% of DM as RDP. Reducing RDP in diets linearly decreased DM intake and tended to decrease milk yield. Milk protein, fat and lactose contents, milk protein yield, body weight, and plasma essential amino acids were unaffected by reduced dietary RDP. However, milk urea-N concentration and milk fat yield decreased linearly with reduced dietary RDP. The apparent efficiency of N utilization for milk N production increased linearly as dietary RDP was reduced. As RDP declined in diets, linear reductions in DM intake and milk production suggested that these cannot be maintained below NRC recommendations of RDP for cows in this study. The aim of the second study was to test the hypothesis that decreasing dietary RDP in lactating dairy cow diets can maintain ruminal metabolism and flow of nutrients out of the rumen and reduce nitrogen excretion. This study was designed as a replicated Latin square with 4 periods of 21 d each. Four treatment diets containing decreasing RDP and constant RUP similar to the first study were used. Three ruminally and duodenally cannulated and 4 ruminally cannulated lactating Holstein cows were randomly assigned to one of the four dietary treatments. A double marker system with Co-EDTA and Yb-labeled forage as markers was used to determine ruminal outflows of nutrients from omasal samples and nutrients reaching the intestine from duodenal samples. Ruminal microbial protein flow was observed using ¹⁵N as an external microbial marker. Feed intake, milk yield, milk composition, and urine and feces output were determined in the last week of each period. Ruminal fluid samples were taken 2 and 4 h after feeding to determine ruminal NH₃-N and volatile fatty acid concentrations. Outflows of nutrients from the rumen were determined by analyzing omasal samples collected over a 24 h feeding cycle in the last week of each period. Reducing dietary RDP decreased protein intakes while DM and fiber intakes were unaffected. Ruminal NH₃-N concentrations linearly declined and peptides and amino acids were unaffected with reduced dietary RDP. A trend for a linear decline in ruminal outflows of microbial N and total N was observed with decreasing dietary RDP. Ruminal volatile fatty acids concentrations were unaltered by feeding treatment diets. Ruminal outflows of DM and acid detergent and neutral detergent fibers were unaffected by treatments. Treatment diets did not have any effect on milk yield and milk composition. However, milk urea-N and milk fat yield decreased linearly with decreasing dietary RDP. Reducing dietary RDP did not affect milk and milk protein yields but did result in greater body protein mobilization. Fecal N output was unaffected however, urine volume and urine N output decreased linearly suggesting reduced environmental N pollution. There was a trend for a linear decrease in total body N balance, but no significant effects on calculated ruminal N balance as dietary RDP decreased. Linear reductions in microbial N leaving the rumen were due to decreased ruminal NH₃-N as peptides plus amino acids and energy supply were unaffected. The linear reduction in milk production and microbial N flow in the first and second studies, respectively, did not support our hypothesis that lactating dairy cows can be fed dietary RDP below current NRC (2001) recommendations without affecting animal performance. The need to raise 15% more cows to alleviate the loss in production may nullify the advantage in reduced N output into the environment by cows fed lower dietary RDP. / Ph. D.

A series of experiments were conducted to investigate how dietary and ruminal factors regulate urea-N recycling in ruminants. In Experiments 1, 2, and 3, urea-N kinetics were measured using 4-d intra-jugular infusions of [15N15N]-urea. In Experiment 1, the objective was to determine how interactions between dietary ruminally-degradable protein (RDP) level and ruminally-fermentable carbohydrate (RFC) may alter urea-N transfer to the gastrointestinal tract (GIT) and the utilization of this recycled urea-N in rapidly-growing lambs fed high N diets. The dietary factors were: 1) dry-rolled barley (DRB) vs. pelleted barley (PB) as the principal source of RFC; and 2) dietary levels of RDP of 60 vs. 70% (% of CP). Nitrogen intake, fecal and urinary N excretion increased as dietary RDP level increased; however, method of barley processing had no effect on N use. Dietary treatment had no effect on urea-N kinetics; however, endogenous production of urea-N (UER) exceeded N intake. For all diets, 0.669 to 0.742 of UER was recycled to the GIT; however, 0.636 to 0.756 of the GER was returned to the ornithine cycle. In Experiment 2, the objective was to delineate the effects of partial defaunation of the rumen on urea-N kinetics in lambs fed low or high N diets. Treatments were: 1) partial defaunation (PDFAUN) vs. faunation (FAUN); and 2) low (10%, LOW) vs. high (15%, HIGH) dietary CP. Linoleic acid-rich sunflower oil was fed as a partially-defaunating agent. Partial defaunation decreased ruminal NH3-N concentrations. The UER and urinary urea-N excretion (UUE) were lower, and the GER tended to be lower in PDFAUN as compared to FAUN lambs; however, as a proportion of UER, GER was higher and the proportion of recycled urea-N that was utilized for anabolism (i.e., UUA) tended to be higher in PDFAUN lambs. The UER, GER and UUE were higher in lambs fed diet HIGH; however, as a proportion of UER, GER and its anabolic use were higher in lambs fed diet LOW. In Experiment 3, the objective was to delineate how, at similar N intakes, interactions between ruminal partial defaunation and altering dietary RFC may alter urea-N kinetics and N metabolism in lambs. Treatments were: 1) PDFAUN vs. FAUN; and 2) DRB vs. PB. Urinary N excretion was lower and retained N was higher in PDFAUN compared to FAUN lambs. The UER was similar across treatments; however, the GER, expressed as absolute amounts or as a proportion of UER, UUA, and microbial N supply were higher in PDFAUN compared to FAUN lambs. As a proportion of UER, GER was higher, whereas UUE was lower in lambs fed PB compared to those fed DRB. In Experiment 4, the objective was to determine the effects of feeding oscillating dietary CP compared to static dietary CP concentration on N retention and in vitro urea flux across ruminal epithelia. Dietary treatments consisted of a medium CP diet (MEDIUM; 12.8% CP) or diets with oscillating CP content (OSC) fed in two different sequences i.e., 2 d of low CP (9.7% CP) followed by 2 d of high CP (16.1% CP; OSC-HIGH) or vice-versa (OSC-LOW). Ruminal epithelial tissues were collected and mounted in Ussing chambers under short-circuit conditions and the serosal-to-mucosal urea flux (Jsm-urea) was measured using 14C-urea. Although N intake was similar, retained N and microbial N supply were greater in lambs fed the OSC diets compared to those fed the MEDIUM diet. The total Jsm-urea was higher in lambs fed the OSC-LOW compared to those fed the OSC-HIGH diet. Across diets, the addition of phloretin (a known specific inhibitor of facilitative urea transporter-B; UT-B) reduced Jsm-urea; however, phloretin-insensitive Jsm-urea was the predominant route for transepithelial urea transfer. In summary, data presented in this thesis provide new insights that the improved N retention typically observed in defaunated ruminants and in ruminants fed oscillating dietary CP concentrations is partly mediated via increased urea-N recycling to the GIT and utilization of recycled urea-N for anabolic purposes.

A series of experiments were conducted to investigate how dietary and ruminal factors regulate urea-N recycling in ruminants. In Experiments 1, 2, and 3, urea-N kinetics were measured using 4-d intra-jugular infusions of [15N15N]-urea. In Experiment 1, the objective was to determine how interactions between dietary ruminally-degradable protein (RDP) level and ruminally-fermentable carbohydrate (RFC) may alter urea-N transfer to the gastrointestinal tract (GIT) and the utilization of this recycled urea-N in rapidly-growing lambs fed high N diets. The dietary factors were: 1) dry-rolled barley (DRB) vs. pelleted barley (PB) as the principal source of RFC; and 2) dietary levels of RDP of 60 vs. 70% (% of CP). Nitrogen intake, fecal and urinary N excretion increased as dietary RDP level increased; however, method of barley processing had no effect on N use. Dietary treatment had no effect on urea-N kinetics; however, endogenous production of urea-N (UER) exceeded N intake. For all diets, 0.669 to 0.742 of UER was recycled to the GIT; however, 0.636 to 0.756 of the GER was returned to the ornithine cycle. In Experiment 2, the objective was to delineate the effects of partial defaunation of the rumen on urea-N kinetics in lambs fed low or high N diets. Treatments were: 1) partial defaunation (PDFAUN) vs. faunation (FAUN); and 2) low (10%, LOW) vs. high (15%, HIGH) dietary CP. Linoleic acid-rich sunflower oil was fed as a partially-defaunating agent. Partial defaunation decreased ruminal NH3-N concentrations. The UER and urinary urea-N excretion (UUE) were lower, and the GER tended to be lower in PDFAUN as compared to FAUN lambs; however, as a proportion of UER, GER was higher and the proportion of recycled urea-N that was utilized for anabolism (i.e., UUA) tended to be higher in PDFAUN lambs. The UER, GER and UUE were higher in lambs fed diet HIGH; however, as a proportion of UER, GER and its anabolic use were higher in lambs fed diet LOW. In Experiment 3, the objective was to delineate how, at similar N intakes, interactions between ruminal partial defaunation and altering dietary RFC may alter urea-N kinetics and N metabolism in lambs. Treatments were: 1) PDFAUN vs. FAUN; and 2) DRB vs. PB. Urinary N excretion was lower and retained N was higher in PDFAUN compared to FAUN lambs. The UER was similar across treatments; however, the GER, expressed as absolute amounts or as a proportion of UER, UUA, and microbial N supply were higher in PDFAUN compared to FAUN lambs. As a proportion of UER, GER was higher, whereas UUE was lower in lambs fed PB compared to those fed DRB. In Experiment 4, the objective was to determine the effects of feeding oscillating dietary CP compared to static dietary CP concentration on N retention and in vitro urea flux across ruminal epithelia. Dietary treatments consisted of a medium CP diet (MEDIUM; 12.8% CP) or diets with oscillating CP content (OSC) fed in two different sequences i.e., 2 d of low CP (9.7% CP) followed by 2 d of high CP (16.1% CP; OSC-HIGH) or vice-versa (OSC-LOW). Ruminal epithelial tissues were collected and mounted in Ussing chambers under short-circuit conditions and the serosal-to-mucosal urea flux (Jsm-urea) was measured using 14C-urea. Although N intake was similar, retained N and microbial N supply were greater in lambs fed the OSC diets compared to those fed the MEDIUM diet. The total Jsm-urea was higher in lambs fed the OSC-LOW compared to those fed the OSC-HIGH diet. Across diets, the addition of phloretin (a known specific inhibitor of facilitative urea transporter-B; UT-B) reduced Jsm-urea; however, phloretin-insensitive Jsm-urea was the predominant route for transepithelial urea transfer. In summary, data presented in this thesis provide new insights that the improved N retention typically observed in defaunated ruminants and in ruminants fed oscillating dietary CP concentrations is partly mediated via increased urea-N recycling to the GIT and utilization of recycled urea-N for anabolic purposes.