Catalyzed phenol-hypochlorite and ninhydrin colorimetric procedures were adapted to the Technicon AutoAnalyzer for simultaneous determination of ammonia and total amino acids in ruminal fluid or ruminal in vitro media. The manifold developed was compatible with a sampling rate of 40/h without significant sample-to-sample carryover. With proper storage, reagents for both the phenol-hypochlorite and the air-stable ninhydrin systems were stable for 8 mo or more. Response of individual amino acids in the phenol-hypochlorite system were generally 1% or less than equimolar amounts of ammonia. Certain amino acids inhibited ammonia color yield 10 to 15% when with equimolar amounts of ammonia; however, the inhibitory effect of casein amino acids was only 2 to 3%. Although ninhydrin response, relative to leucine, of individual alpha-amino acids ranged from 62 (valine) to 151% (histidine), recoveries of casein amino acids from ruminal fluid had coefficients of variation of 1% or less. Coefficients of variation for ammonia recoveries from ruminal fluid by the phenol-hypochlorite procedure were about half of those for the Conway microdiffusion technique. Intraclass correlations for the adapted procedures indicated high degrees of accuracy and precision for both ammonia and amino acid analyses.

Twenty-four multiparous dairy cows (eight with ruminal cannulae) were blocked by days in milk and assigned to six balanced 4 x 4 Latin squares with 21-d periods. The four diets, formulated from alfalfa silage plus a concentrate mix based on ground high moisture ear corn, contained (dry matter basis): 1) 20% concentrate, 80% alfalfa silage (24% nonfiber carbohydrate; NFC), 2) 35% concentrate, 65% alfalfa silage (30% NFC), 3) 50% concentrate, 50% alfalfa silage (37% NFC), or 4) 65% concentrate, 35% alfalfa silage (43% NFC). Soybean meal and urea were added to make diets isonitrogenous with equal nonprotein nitrogen (NPN) (43% of total N). Total urine was collected with indwelling Folley catheters for 24 h during each period. There was no effect of diet on urinary creatinine excretion (average 29 mg/kg of BW/d). There were quadratic effects of diet on total urinary ecretion of allantoin, uric acid, and purine derivatives (allantoin plus uric acid), and on ruminal synthesis of microbial N estimated from purine derivatives; maxima occurred at about 35% dietary NFC. Urinary excretion also was estimated with spot urine samples from creatinine concentration and the mean daily creatinine excretion. Daily excretion of allantoin, uric acid, and purine derivatives estimated from spot urine sampling followed the same pattern as that observed with total collection; differences between measured and estimated urine volume were significant only for 35% dietary concentrate. Spot urine sampling appeared to yield satisfactory estimates of purine derivative excretion. Maximal urea N excretion was estimated to occur at about 31% dietary NFC. Milk allantoin secretion increased linearly with concentrate and accounted for 4 to 6% of the total purine derivative excretion. Microbial yield was maximal at 35% dietary NFC, suggesting that this was the optimal level for utilization of dietary NPN from alfalfa silage and other sources.

Forty-five multiparous and 18 primiparous Holstein cows were fed three levels of crude protein (CP), each at three levels of neutral detergent fiber (NDF), to identify optimal dietary CP and energy. Cows were blocked by parity and days in milk into seven groups of nine and randomly assigned to an incomplete 9 x 9 Latin square trial with four, 4-wk periods. Diets were formulated from alfalfa and corn silages, high-moisture corn, soybean meal, minerals, and vitamins. Forage was 60% alfalfa and 40% corn silage on all diets; NDF contents of 36, 32, and 28% were obtained by feeding 75, 63, and 50% forage, respectively. Dietary CP contents of 15.1, 16.7, and 18.4% were obtained by replacing high-moisture corn with soybean meal. Production data were from the last 2 wk of each period. Spot fecal and urine samples were collected from 36 cows to estimate N excretion using fecal indigestible acid detergent fiber (ADF) and urinary creatinine as markers. There were no interactions (P > or = 0.08) between dietary CP and NDF for any trait; thus, effects of CP were not confounded by NDF or vice versa. Intake of DM and fat yield were lower on 15.1% CP than at higher CP. There were linear increases in milk urea and urinary N excretion and linear decreases in N efficiency with increasing CP. Increasing CP from 15.1 to 18.4% reduced milk N from 31 to 25% of dietary N, increased urinary N from 23 to 35% of dietary N, and reduced fecal N from 45 to 41% of dietary N. Decreasing NDF gave linear increases in BW gain, yield of milk, protein, true protein, lactose, and SNF, and milk/DM intake and milk N/N intake, and linear decreases in milk urea. However, fat yield was lower on 28% than 32% NDF. Reducing NDF from 36 to 28% increased purine derivative excretion by 19%, suggesting increased microbial protein. Increasing CP by adding soybean meal to diets fed cows averaging 34 kg/d of milk increased intake and fat yield but depressed N efficiency. Increasing dietary energy by reducing forage improved milk yield and efficiency and decreased excretion of environmentally labile urinary N.

Data from 35 trials with 482 lactating cows fed 106 diets were used to study the effects of animal and dietary factors on the relationship between milk and blood urea N and the value of milk urea N in the assessment of protein status. In two trials, urea N in whole blood and in blood plasma were closely related (r2 = 0.952); the slope was not significantly different from 1.0, and the intercept was not significantly different from 0. Regression of milk urea N on blood urea N with a mixed effects model using all 2231 observations yielded the equation: milk urea N (milligrams of N per deciliter) = 0.620 x blood urea N (milligrams of N per deciliter) + 4.75 (r2 = 0.842); this model accounted for a significant interaction of cow and blood urea N. Single factors that yielded significant regressions on milk urea N with the mixed effects models were dietary crude protein (CP) (percentage of dry matter; r2 = 0.839), dietary CP per megacalorie of net energy for lactation (NEL) (r2 = 0.833), excess N intake (r2 = 0.772), N efficiency (r2 = 0.626), and ruminal NH3 (r2 = 0.574). When all factors were analyzed at once, 12 were significant in a mixed effects model. Blood urea N, body weight, yield of fat-corrected milk, dietary CP content, excess N intake, dry matter intake, and days in milk were positively related to milk urea N, and parity, milk and fat yield, dietary CP per unit of NEL content, and NEL intake were negatively related to milk urea N. In one trial, the mean urea concentration was 35 times greater in urine than in milk; lower proportions of total urea excretion in milk were observed in the a.m. sampling (1.8%) than in the p.m. sampling (3.3%). Measuring urea N in a composite milk sample from the whole day substantially improved reliability of data. The number of cows fed a specific diet that must be sampled to determine mean milk urea N within 95% confidence intervals with half widths of 1.0 and 2.0 mg of N/dl was estimated to be 16 and 4, respectively.

Forty lactating Holstein cows, including 10 with ruminal cannulas, were blocked by days in milk into 8 groups and then randomly assigned to 1 of 8 incomplete 5 x 5 Latin squares to assess the effects of 5 levels of dietary crude protein (CP) on milk production and N use. Diets contained 25% alfalfa silage, 25% corn silage, and 50% concentrate, on a dry matter (DM) basis. Rolled high-moisture shelled corn was replaced with solvent-extracted soybean meal to increase CP from 13.5 to 15.0, 16.5, 17.9, and 19.4% of DM. Each of the 4 experimental periods lasted 28 d, with 14 d for adaptation and 14 d for data collection. Spot sampling of ruminal digesta, blood, urine, and feces was conducted on d 21 of each period. Intake of DM was not affected by diet but milk fat content as well as ruminal acetate, NH3, and branched-chain volatile fatty acids, urinary allantoin, and blood and milk urea all increased linearly with increasing CP. Milk and protein yield showed trends for quadratic responses to dietary CP and were, respectively, 38.3 and 1.18 kg/d at 16.5% CP. As a proportion of N intake, urinary N excretion increased from 23.8 to 36.2%, whereas N secreted in milk decreased from 36.5 to 25.4%, as dietary protein increased from 13.5 to 19.4%. Under the conditions of this study, yield of milk and protein were not increased by feeding more than 16.5% CP. The linear increase in urinary N excretion resulted from a sharp decline in N efficiency as dietary CP content increased.

Replacing dietary starch with sugar has been reported to improve production in dairy cows. Two sets of 24 Holstein cows averaging 41 kg/d of milk were fed a covariate diet, blocked by days in milk, and randomly assigned in 2 phases to 4 groups of 6 cows each. Cows were fed experimental diets containing [dry matter (DM) basis]: 39% alfalfa silage, 21% corn silage, 21% rolled high-moisture shelled corn, 9% soybean meal, 2% fat, 1% vitamin-mineral supplement, 7.5% supplemental nonstructural carbohydrate, 16.7% crude protein, and 30% neutral detergent fiber. Nonstructural carbohydrates added to the 4 diets were 1) 7.5% corn starch, 0% sucrose; 2) 5.0% starch, 2.5% sucrose; 3) 2.5% starch, 5.0% sucrose; or 4) 0% starch, 7.5% sucrose. Cows were fed the experimental diets for 8 wk. There were linear increases in DM intake and milk fat content and yield, and linear decreases in ruminal concentrations of ammonia and branched-chain volatile fatty acids, and urinary excretion of urea-N and total N, and urinary urea-N as a proportion of total N, as sucrose replaced corn starch in the diet. Despite these changes, there was no effect of diet on microbial protein formation, estimated from total purine flow at the omasum or purine derivative excretion in the urine, and there were linear decreases in both milk/DM intake and milk N/N-intake when sucrose replaced dietary starch. However, expressing efficiency as fat-corrected milk/DM intake or solids-corrected milk/DM intake indicated that there was no effect of sucrose addition on nutrient utilization. Replacing dietary starch with sucrose increased fat secretion, apparently via increased energy supply because of greater intake. Positive responses normally correlated with improved ruminal N efficiency that were altered by sucrose feeding were not associated with increased protein secretion in this trial.

Adding sugar to the diet has been reported to improve production in dairy cows. In each of 2 trials, 48 lactating Holsteins (8 with ruminal cannulas) were fed covariate diets for 2 wk, blocked by days in milk into 12 groups of 4, and then randomly assigned to diets based on alfalfa silage containing 4 levels of dried molasses (trial 1) or liquid molasses (trial 2). In both studies, production data were collected for 8 wk, ruminal samples were taken in wk 4 and 8, and statistical models were used that included covariate means and block. In trial 1, experimental diets contained 18% CP and 0, 4, 8, or 12% dried molasses with 2.6, 4.2, 5.6, or 7.2% total sugar. With increasing sugar, there was a linear increase in dry matter intake (DMI), and digestibility of dry matter (DM) and organic matter (OM), but no effect on yield of milk or protein. This resulted in linear decreases in fat-corrected milk (FCM)/DMI and milk N/N-intake. There was a linear decrease in urinary N with increasing sugar, and quadratic effects on milk fat content, yield of fat and FCM, and ruminal ammonia. Mean optimum from these quadratic responses was 4.8% total sugar in these diets. In trial 2, experimental diets contained 15.6% crude protein (CP) and 0, 3, 6, or 9% liquid molasses with 2.6, 4.9, 7.4, or 10.0% total sugar, respectively. Again, there were linear declines in FCM/DMI and milk N/N-intake with increasing sugar, but quadratic responses for DMI, yield of milk, protein, and SNF, digestibility of neutral detergent fiber and acid detergent fiber, milk urea, urinary excretion of purine derivatives, and ruminal ammonia. Mean optimum from all quadratic responses in this trial was 6.3% total sugar. An estimate of an overall optimum, based on yield of fat and FCM (trial 1) and yield of milk, protein, and SNF (trial 2), was 5.0% total sugar, equivalent to adding 2.4% sugar to the basal diets. Feeding more than 6% total sugar appeared to depress production.

Twenty-eight (8 with ruminal cannulas) lactating Holstein cows were assigned to 4 × 4 Latin squares and fed diets with different levels of rumen-degraded protein (RDP) to study the effect of RDP on production and N metabolism. Diets contained [dry matter (DM) basis] 37% corn silage, 13% alfalfa silage, and 50% concentrate. The concentrate contained solvent and lignosulfonate-treated soybean meal and urea, and was adjusted to provide RDP at: 13.2, 12.3, 11.7, and 10.6% of DM in diets A to D, respectively. Intake of DM and yield of milk, fat-corrected milk, and fat were not affected by treatments. Dietary RDP had positive linear effects on milk true protein content and microbial nonammonia N (NAN) flow at the omasal canal, and a quadratic effect on true protein yield, with maximal protein production at 12.3% RDP. However, dietary RDP had a positive linear effect on total N excretion, with urinary N accounting for most of the increase, and a negative linear effect on environmental N efficiency (kg of milk produced per kg of N excreted). Therefore, a compromise between profitability and environmental quality was achieved at a dietary RDP level of 11.7% of DM. Observed microbial NAN flow and RDP supply were higher and RUP flow was lower than those predicted by the NRC ( 2001) model. The NRC ( 2001) model overpredicted production responses to RUP compared with the results in this study. Replacing default NRC degradation rates for protein supplements with rates measured in vivo resulted in similar observed and predicted values, suggesting that in situ degradation rates used by the NRC are slower than apparent rates in this study.