Form of Rumen-Degradable Carbohydrate and Nitrogen on Microbial Protein Synthesis and Protein Efficiency of Dairy Cows1

J. Dairy Sci. 85:900–908  American Dairy Science Association, 2002.

Form of Rumen-Degradable Carbohydrate and Nitrogen on Microbial Protein Synthesis and Protein Efficiency of Dairy Cows1 R. A. Sannes,*,† M. A. Messman,‡ and D. B. Vagnoni*,‡ *Animal, Dairy, and Veterinary Sciences, Utah State University, Logan 84322 †Present address: School of Veterinary Medicine, University of Wisconsin, Madison 53706 ‡Present address: Cargill Animal Nutrition Center, Elk River, MN 55360

ABSTRACT Sixteen multiparous lactating Holstein cows (four with rumen cannulae) were fed diets varying in the content and form of ruminally degradable carbohydrates and N to examine dietary effects on microbial protein synthesis (MPS) and whole animal N efficiency, and to evaluate the use of a model based on milk urea N (MUN) for predicting urinary N excretion and N utilization efficiency (NUE). A replicated Latin square design (consisting of diet and experimental period) was employed. The four diets consisted of two low protein diets with either 20% ground corn (diet LP) or 13.5% ground corn plus 3% sucrose (diet LP sucrose) and two high protein diets with 13.5% corn and 3% sucrose with either urea (diet HP urea) or soybean meal (diet HP SBM) as supplemental rumen-degradable protein sources. The intakes of dry matter and N were increased by increasing dietary crude protein (CP) level. However, the yields of milk and milk protein were not affected by CP level. Yield of microbial protein was reduced by sucrose and increased by CP level. There were no differences between urea and SBM supplementation on DM intake, milk yield, or MPS. Mean urinary N excretion for all cows (252 g/d) was underestimated by 55 g/d or overestimated by 25 or 33 g/d using alternative equations based on MUN. Subsequently, NUE (mean = 22.4%) was underestimated by 7.5, 3.2, or 2.9%, using a previously published set of equations. Urinary N excretion and NUE could be predicted within 10 and 14% of observed values, respectively, using a set of equations incorporating MUN. Therefore, MUN appears to be a useful tool to help assess N losses from lactating cows. (Key words: carbohydrate, degradable nitrogen, microbial protein synthesis, dairy)

Received July 9, 2001. Accepted November 16, 2001. Corresponding author: D. B. Vagnoni; e-mail: david_vagnoni@ cargill.com. 1 Published as Utah State Agricultural Experiment Station journal paper number 7399.

Abbreviation key: BUN = blood urea nitrogen, DTP = degradable true protein, LP = low protein, HP = high protein, MPS = microbial protein synthesis, MUN = milk urea nitrogen, NUE = N utilization efficiency, PD = purine derivatives, SBM = soybean meal. INTRODUCTION Ruminant animals use dietary CP relatively inefficiently, due largely to ruminal N metabolism (Broderick et al., 1991). High producing dairy cows are fed large quantities of high quality proteins, and, because microbial protein degradation is not directly coupled to microbial protein synthesis (MPS), ruminal NH3 production often is excessive and ultimately lost by urinary N excretion. Increasing the supply of ruminally fermentable starch by either increasing the dietary content of high moisture corn (Vagnoni and Broderick, 1997) or finely grinding high moisture corn (Ekinci and Broderick, 1997) reduced ruminal NH3 concentrations and increased milk protein yield (Ekinci and Broderick, 1997; Vagnoni and Broderick, 1997), probably through increased MPS (Vagnoni and Broderick, 1997). The addition of sugars also has increased ruminal and wholeanimal N efficiency of sheep and steers, reportedly to a greater extent than starch (Chamberlain and Thomas, 1983; Chamberlain et al., 1993; Chamberlain and Choung, 1995). The form of ruminally degradable N also has an effect on microbial growth and protein production. There is substantial evidence for enhanced MPS from the provision of degradable true protein (DTP) as peptides and amino acids to bacteria grown on soluble carbohydrates in vitro (Cotta and Russell, 1982; Argyle and Baldwin, 1989; Cruz Soto et al., 1994), however, in vivo responses to DTP have been less consistent (Armentano et al., 1993; Chikunya et al., 1996). Urinary urea excretion is proportional to blood urea concentration (Ciszuk and Gebregziabher, 1994). Because milk urea N (MUN) is closely related to blood urea N (BUN) (Roseler et al., 1993), Jonker et al. (1998) proposed a model that integrated the use of MUN for predicting urinary N excretion and N efficiency of lac-

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tating cows. The objectives of this study were: 1) to evaluate the effects of dietary sugar versus starch and DTP versus NPN on MPS and N utilization efficiency (NUE) and 2) to evaluate the model of Jonker et al. (1998) for predicting N excretion and NUE in lactating cows. MATERIALS AND METHODS Cows and Feeding Sixteen multiparous Holstein cows (four with ruminal cannulae) with 690 ± 67 (mean ± SD) kg of BW and 107 ± 27 DIM were assigned to four replicated 4 × 4 Latin squares with 21-d periods. Two low protein diets were formulated to be 17% CP and contained either 20% corn (diet LP) or 13.5% corn plus 3.2% sucrose

(diet LP sucrose). Additionally, two high protein diets were formulated to be 18.5% CP and contained a similar level of corn and sucrose as diet LP sucrose, but contained additional urea (diet HP urea) or soybean meal (diet HP SBM) as supplemental sources of RDP. Diets were formulated to be isoenergetic and equalized for RUP content within CP level. Animals were blocked by cannulation and DIM. Days 1 to 14 served as the adaptation period and all samples and data were collected from d 15 to 21. Diets (Table 1) were fed for ad libitum intake twice daily as TMR to allow for 10% orts. A forage mixture of alfalfa hay, corn silage, and whole cottonseeds was prepared daily and blended with the concentrate ingredients to form the TMR. The content of the forage mixture (as-fed basis) was adjusted as needed based on the corn silage DM, which was deter-

Table 1. Ingredient and nutrient composition of the diets (% of DM).1 Ingredient

LP

LP sucrose

HP urea

HP SBM

Corn silage Alfalfa hay Whole cottonseeds Ground corn Wheat midds Soy hulls Blood meal Corn gluten meal Beet pulp Distiller’s dried grains Feather meal Urea Soybean meal Sucrose Fat Salt Limestone Dicalcium phosphate Sodium bicarbonate Magnesium oxide Trace-mineralized premix2 Dynamate3 Vitamin premix4 Se premix5 Total Nutrient DM CP RUP6 NEL, Mcal/kg NDF ADF Ca P

33.00 25.40 3.60 19.86 0.84 1.68 1.18 2.86 5.05 3.37 0.00 0.30 0.00 0.00 0.59 0.07 0.34 0.81 0.50 0.39 0.05 0.03 0.05 0.04 100.00

33.00 25.40 3.60 13.53 2.03 1.86 1.18 2.88 5.07 5.07 0.00 0.29 0.00 3.21 0.59 0.07 0.34 0.81 0.50 0.39 0.05 0.03 0.06 0.04 100.00

33.00 25.40 3.60 13.72 3.33 1.71 1.03 3.09 5.14 2.57 0.51 0.77 0.00 3.43 0.60 0.07 0.26 0.68 0.50 0.39 0.05 0.03 0.06 0.04 100.00

33.00 25.40 3.60 13.40 1.00 1.67 1.00 2.68 5.03 1.67 0.00 0.42 5.36 3.02 0.59 0.09 0.33 0.67 0.50 0.39 0.05 0.03 0.05 0.04 100.00

53.0 17.4 37.0 1.61 34.0 22.6 0.78 0.50

52.9 17.0 36.0 1.61 34.3 22.8 0.78 0.52

53.6 18.5 33.0 1.60 34.4 22.8 0.73 0.47

54.1 19.6 33.0 1.62 34.0 22.6 0.76 0.46

1 LP = Low protein diet; LP sucrose = low protein diet plus sucrose; HP urea = high protein diet containing urea; HP SBM = high protein diet containing soybean meal. 2 Provided (per kg of DM): Mn, 60 mg; Cu, 11 mg; Zn, 81 mg; Fe, 27 mg; Co, 1 mg; I, 1 mg. 3 Magnesium and potassium sulfate (Marshall Minerals, Marshall, TX). 4 Provided (per kg of DM): vitamin A, 6680 IU; vitamin D, 1550 IU; and vitamin E, 21 IU. 5 Provided (per kg of DM) 0.2 mg of Se. 6 Computed from published values (National Research Council, 1989) for forages and analyzed values for concentrates and their dietary proportions.

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SANNES ET AL. Table 2. Least squares means for ruminal pH and metabolite concentrations. Contrast2, P > F3

Diet1 Item

LP

LP sucrose

HP urea

HP SBM

SE

Sucrose

CP level

CP source

NH3, mM Total VFA, mM Acetate, mM Propionate, mM Butyrate, mM Total BCVFA4, mM

4.92 131.03 85.34 26.34 16.19 1.87

3.89 123.23 77.81 27.08 15.53 1.34

6.45 125.98 81.91 24.80 16.41 1.58

9.07 133.7 87.29 25.90 16.88 2.10

0.35 6.09 3.21 2.02 0.97 0.12

0.082 0.401 0.149 0.805 0.650 0.018

<0.001 0.410 0.135 0.512 0.385 0.012

0.002 0.405 0.281 0.713 0.742 0.021

1 LP = Low protein diet; LP sucrose = low protein diet plus sucrose; HP urea = high protein diet containing urea; HP SBM = high protein diet containing soybean meal. 2 Sucrose: LP vs. LP sucrose; CP level: LP sucrose vs. HP urea and HP SBM; CP source: HP urea vs. HP SBM. 3 Probability of a significant contrast effect. 4 Branched chain volatile fatty acids.

mined weekly at 60°C (48 h). Four separate concentrates, one for each diet, were prepared from the remaining ingredients and used throughout the experiment. Weekly composites of the forage mixture, corn silage, whole cottonseed, concentrates, TMR, and orts were prepared from daily samples of approximately 0.5 kg stored at −20°C. Cows had free access to water throughout the trial. Milk yield was recorded at both the a.m. and p.m. milkings. Sampling

served with the addition of 1 ml of 50% (vol/vol) H2SO4 per 50 ml of ruminal fluid and stored at −20°C. Additional ruminal samples (1 L) were obtained at 0, 1, 4, 8, and 12 h and preserved with formalin (50-ml of formalin/L of ruminal contents). These samples were composited, stored at 4°C for approximately 12 to 24 h, and then squeezed through two layers of cheesecloth and washed three times with a total of 3 L of McDougall’s buffer (McDougall, 1948). Ruminal fluid plus buffer was centrifuged (550 × g at 4°C, 10 min), the supernatants were decanted, and centrifuged (15,000

Total urine collections were made using indwelling Foley catheters, inserted on d 18 of each experimental period. Urinary output was measured every 24 h for 3 d. Fresh containers with 1 L of 50% (vol/vol) H2SO4 were attached to each catheter after p.m. milkings (final pH < 3). Just before milking, catheters were clamped shut, and cows were led to the milking parlor, milked, and immediately returned to their stalls; fresh containers were then attached. Catheters remained clamped for approximately 30 min at each milking. After the weight of the urine was recorded, its specific gravity was determined with a clinical refractometer. Urinary volume was computed as the quotient of urinary weight and specific gravity. Two-milliliter aliquots of urine were diluted to 20 ml with tap water and stored at −20°C. Milk samples also were obtained at the a.m. and p.m. milkings for the last 3 d of each experimental period. Whole rumen contents were obtained from the cannulated cows at 0, 1, 2, 4, 6, 8, and 12 h post-a.m. feeding on the last day of each experimental period. Samples (consisting of 200 ml of subsamples from five locations in the rumen) were strained through two layers of cheesecloth, and the pH of the strained rumen fluid was determined using a combination electrode (Orion model 370). Subsamples of ruminal fluid were then pre-

Figure 1. Hourly mean values of ruminal NH3 concentrations. Pooled SE of diet by time means equals 1.10.

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PROTEIN EFFICIENCY OF LACTATING COWS Table 3. Least squares means for DMI, milk yield, and the composition and yield of milk components. Contrast2, P > F3

Diet1 Item

LP

LP sucrose

HP urea

HP SBM

SE

Sucrose

CP level

CP source

DMI, kg/d Milk, kg/d Fat, % Protein, % Lactose, % SNF, % Yield, kg/d Fat Protein Lactose

25.73 34.34 3.88 3.14 4.77 8.65

25.54 33.20 3.83 3.12 4.76 8.63

26.82 33.64 3.88 3.10 4.74 8.58

26.27 33.72 3.87 3.13 4.76 8.62

0.29 0.32 0.05 0.02 0.02 0.03

0.644 0.018 0.556 0.570 0.487 0.585

0.008 0.239 0.512 0.655 0.609 0.485

0.192 0.855 0.897 0.391 0.467 0.373

1.33 1.07 1.64

1.27 1.03 1.58

1.30 1.04 1.59

1.31 1.05 1.60

0.02 0.01 0.02

0.043 0.046 0.028

0.162 0.569 0.469

0.870 0.688 0.761

1 LP = Low protein diet; LP sucrose = low protein diet plus sucrose; HP urea = high protein diet containing urea; HP SBM = high protein diet containing soybean meal. 2 Sucrose: LP vs. LP sucrose; CP level: LP sucrose vs. HP urea and HP SBM; CP source: HP urea vs. HP SBM. 3 Probability of a significant contrast effect.

× g at 4°C, 20 min), and the resulting bacterial pellets were dried at 60°C for 48 h. Blood samples were taken 4 h post-a.m. feeding on the last day of each period from the coccygeal artery or vein. Samples were collected into heparinized glass tubes, held on ice for several hours, and then spun to harvest plasma. Plasma was deproteinized by adding one volume of plasma to one volume of 10% TCA. Laboratory Analyses Weekly composites of the feed ingredients, forage mixture, concentrates, TMR, and orts were dried at 60°C for 48 h, ground, and then analyzed for N (CE Elantech NA2100 N analyzer), DM (105°C for 24 h), and NDF (including sodium sulfite and amylase) and ADF (Robertson and Van Soest, 1981). Concentrate samples were analyzed for RUP at Dairy One (Ithaca, NY) using a procedure based on Streptomyces griseus protease in borate buffer (Roe et al., 1990). Dietary RUP values were computed using published values for the forages (National Research Council, 1989) and the analyzed values for the concentrates and the respective dietary proportions. Daily milk composites were constructed from the a.m. and p.m. milk samples, weighted according to the respective a.m. and p.m. milk yields. Milk samples then were split. One subsample was analyzed for protein, fat, SNF, lactose, SCC, and MUN at the Rocky Mountain DHIA Laboratory (Logan, UT). The other subsample was treated with TCA (1:1 dilution with 25% TCA), filtered through #4 Whatman paper and the supernatant was stored frozen for later analysis of uric acid (Sigma kit 685), and allantoin (Chen et al., 1993). Urine was analyzed for total N by adding 100 µl of 1/10 diluted urine to 8- × 5-mm tin capsules (CE Instruments, Ther-

moQuest, Milan, Italy) containing a small quantity of Chromasorb (Elemental Microanalysis Limited) absorbent. Samples were dried overnight at 40°C and then analyzed for total N (CE Elantech NA2100 N analyzer). Urine was also analyzed for uric acid (Sigma kit 685), allantoin (Chen et al., 1993), and urea N (Crocker, 1967). Plasma was analyzed for urea N (Crocker, 1967). Acidified ruminal fluid was thawed, centrifuged (30,000 × g at 4°C, 15 min), and analyzed for NH3 (Chaney and Marbach, 1962). Areas under time-course curves for ruminal pH and NH3 were calculated using the trapezoidal rule, and mean concentrations were computed by dividing areas by the number of hours (12 h). To reduce the number of VFA determinations, cow × period composites of ruminal fluid were prepared and analyzed (Weimer et al., 1991). Total VFA concentrations were computed as the sum of acetate, propionate, butyrate, valerate, and the branched-chain VFA (isovalerate, 2methyl butyrate, and isobutyrate). Dried bacterial samples were ground in a coffee grinder and analyzed for total N (CE Elantech NA2100 nitrogen N) and for purines by HPLC (Vagnoni et al., 1997). The yield of MPS was estimated using the following equation: MPS (g/d) = (g of microbial CP/mmol of purine) × [(mmol of PD excreted per day − 103)/0.856] (Vagnoni et al., 1997). Statistical Analyses Data were analyzed as a 4 × 4 Latin square, replicated once for ruminal data and replicated four times for all other responses, using the general linear models procedure of SAS (1996). The model for the replicated Latin square included square, cow within square, period, diet, period × square, and diet × square. Interactions (P ≥ 0.14) were not significant and were pooled with the residual. The model for the ruminal Latin Journal of Dairy Science Vol. 85, No. 4, 2002

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Table 4. Least squares means for parameters of N metabolism. Contrast2, P > F3

Diet1 Item

LP

LP sucrose

HP urea

HP SBM

SE

Sucrose

CP level

CP source

Urinary volume, L/d Total urinary N, g/d Urinary urea N, g/d Urinary urea N/total urinary N Milk urea N, mg/dl Blood urea N, mg/dl NUE4

22.5 233.9 165.3 0.72 14.3 13.9 0.23

21.8 209.6 144.0 0.71 13.3 13.8 0.23

25.4 274.0 199.3 0.74 16.6 15.8 0.20

25.7 289.3 227.5 0.83 18.7 19.5 0.19

0.55 10.5 6.0 0.02 0.3 0.9 0.005

0.400 0.110 0.015 0.765 0.057 0.950 0.574

<0.001 <0.001 <0.001 0.002 <0.001 <0.001 <0.001

0.672 0.309 0.002 0.003 <0.001 0.004 0.136

1 LP = Low protein diet; LP sucrose = low protein diet plus sucrose; HP urea = high protein diet containing urea; HP SBM = high protein diet containing soybean meal. 2 Sucrose: LP vs. LP sucrose; CP level: LP sucrose vs. HP urea and HP SBM; CP source: HP urea vs. HP SBM. 3 Probability of a significant contrast effect. 4 Nitrogen utilization efficiency.

square consisted of cow, period, and diet. Mean separations were performed using the following set of preplanned single degree-of-freedom contrasts: 1) sucrose (diet LP vs. diet LP sucrose), 2) CP level (diet LP sucrose vs. diet HP urea and diet HP SBM), and 3) CP source (diet HP urea vs. diet HP SBM). RESULTS AND DISCUSSION Crude protein was similar in the low protein diets (average of 17.2%), and higher in high protein diets (average of 19.1%), as intended (Table 1). However, the CP content was higher than intended in diet HP SBM (19.6%); diets were intended to be isonitrogenous within CP levels. Dietary RUP levels were similar within the low protein (average = 36.5%) and high protein (average = 33.0%) as intended. Neutral detergent fiber and ADF values were similar across dietary treatments. There was no dietary effect (P ≥ 0.478) on ruminal pH (average = 6.02). Sucrose tended to reduce (P = 0.082) mean ruminal NH3 concentration (Table 2). Chamberlain et al. (1985) reported a reduction in ruminal NH3 concentration due to sucrose supplementation of a grass silage diet and found sucrose to be more effective than starch in reducing ruminal NH3 concentrations. Mean ruminal NH3 concentrations were increased (P < 0.001) by increasing CP level and were higher (P = 0.002) in diets containing SBM versus urea. Higher ruminal NH3 concentrations for SBM versus

urea were surprising and very likely due to the higher CP content of the HP SBM diet. The mean NH3 concentrations on all diets remained above the value of 3.5 mM suggested (Satter and Slyter, 1974) as the minimum necessary for maintenance of ruminal bacterial growth. However, hourly means reveal that concentrations fell below this level after 4 h postfeeding for diet LP sucrose and after 8 h postfeeding for diet LP (Figure 1). Hourly means never dropped below this level for high protein diets. Therefore, differences observed between low protein and high protein diets may partially reflect a response due to ruminal NH3 limitation. There were no dietary effects (P ≥ 0.135) on the concentrations of total VFA, acetate, propionate, or butyrate. Concentrations of VFA represent a balance between production and disappearance, and differences in production rate may not be apparent from VFA concentrations (Leng, 1970). Total branched chain VFA concentrations were reduced (P = 0.018) by sucrose and were increased with higher CP levels (P = 0.012) and for diets containing SBM vs. urea (P = 0.021). The branched chain VFA are produced in the rumen from the deamination and decarboxylation of the branch-chained amino acids (Allison, 1970). Ruminal branched-chain amino acids may arise from feed protein or microbial protein, and differences in branched chain VFA concentrations probably reflect differences in one or both of these components. Daily DMI was unaffected by sucrose or CP source (P ≥ 0.192), but was increased (P = 0.008) by increased

Table 5. Coefficients of determination among dietary means of some parameters of N metabolism.

Urinary N Urinary urea N NUE1 Ruminal NH3 1

Nitrogen utilization efficiency.

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MUN

BUN

Ruminal NH3

0.955 0.995 0.944 0.982

0.775 0.886 0.846 0.961

0.895 0.971 0.878 ...

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PROTEIN EFFICIENCY OF LACTATING COWS Table 6. Least squares means for purine derivative (PD) excretion and microbial protein synthesis. Contrast2, P > F3

Diet1 Item

LP

LP sucrose

HP urea

HP SBM

SE

Sucrose

CP level

CP source

Total PD excretion, mmol/d Bacterial CP:purines, g/mmol Microbial CP, g/d

522.2 4.21 2091

500.4 3.88 1830

527.2 3.98 1981

538.8 4.24 2133

17.4 0.16 80

0.380 0.208 0.026

0.133 0.296 0.025

0.639 0.303 0.184

1 LP = Low protein diet; LP sucrose = low protein diet plus sucrose; HP urea = high protein diet containing urea; HP SBM = high protein diet containing soybean meal. 2 Sucrose: LP vs. LP sucrose; CP level: LP sucrose vs. HP urea and HP SBM; CP source: HP urea vs. HP SBM. 3 Probability of a significant contrast effect.

CP level (Table 3). Milk yield was decreased (P = 0.018) by sucrose but was unaffected (P ≥ 0.239) by other dietary factors. There were no dietary effects (P ≥ 0.373) on milk composition, but the yield of all milk components was decreased by sucrose (P ≤ 0.046). The increased DMI observed with the high protein diets may have been due to the increased ruminal NH3 concentration associated with these diets (Table 2 and Figure 1). Insufficient ruminal NH3 may depress microbial function (e.g., growth, fiber digestion) and thereby limit intake. However, when ruminal NH3 was adequate, as was the case for the high protein diets, intakes were similar whether or not the additional RDP was provided as NPN or DTP. Our intention was for cows receiving LP diets to be limited for milk protein yield relative to cows receiving HP diets, and to use milk protein responses obtained with diets HP urea and HP SBM

to evaluate NPN versus DTP. However, milk production obtained in this experiment was lower than expected, and we were unable to make this evaluation (i.e., yield of milk protein was unaffected by CP level). Sucrose tended to reduce (P ≤ 0.110) total urinary N excretion and MUN and reduced (P = 0.015) urinary urea N excretion (Table 4). These responses are consistent with the reduced ruminal NH3 caused by sucrose (Table 2, Figure 1) and the observations of others, including decreased plasma urea N concentration and urinary urea N excretion in sheep (Obara and Dellow, 1993) and increased transfer of BUN to the rumen in sheep (Kennedy et al., 1981). These effects suggest a potential for improved NUE, but sucrose had no effect (P = 0.574) on NUE. Urinary volume was increased (P < 0.001) by the high protein diets, probably due to the limited ability of the

Table 7. Prediction of N Metabolism using equations based on MUN (Jonker et al., 1998).

Prediction

1

Original MUN equation UN g/d NI g/d NUE, % DMI, kg/d

Mean bias

Actual mean

Prediction mean

RMSPE

251.7 749.2 22.38 25.81

197.1 553.2 29.91 19.08

56.2 66.5 2.06 1.87

251.73 749.2 22.38 25.81

276.7 649.0 25.57 22.37

251.73 749.2 22.38 25.81

284.4 658.4 25.24 22.69

2

Linear bias

P

Bias4

P

54.6 196.0 −7.53 6.73

<.001 <.001 <.001 <.001

−0.384 −0.093 −0.146 −0.850

0.048 0.586 0.041 <0.001

56.2 64.8 2.12 1.86

−24.9 100.2 −3.20 3.44

.002 <.001 <.001 <.001

−0.562 −0.276 −0.139 −0.835

<0.001 0.030 0.064 <0.001

53.5 65.1 2.32 1.81

−32.7 90.8 −2.86 3.11

<.001 <.001 <.001 <.001

−0.509 −0.389 −0.134 −0.778

<0.001 <0.001 0.121 <0.001

Bias

3

5

Modified MUN equation6 UN g/d NI g/d NUE, % DMI, kg/d Modified MUN equation7 UN g/d NI g/d NUE, % DMI, kg/d

UN = Urinary N excretion; NI = N intake; NUE = N utilization efficiency. RMSPE = Root mean square prediction error. 3 Mean observation − mean prediction. 4 Slope of the line from regression of residuals on predicted values. 5 UN = 12.54 × MUN (mg/dl) (Jonker et al., 1998). 6 UN = 17.6 × MUN (mg/dl) (Kauffman and St.-Pierre, 1999). 7 UN = 0.026 × BW (kg) × MUN (mg/dl) (R. A. Kohn, 2001, personal communication). 1 2

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kidneys to concentrate the increased urea N excreted in these diets (Table 4). Total urinary N, urinary urea N, MUN, and BUN also were increased (P ≤ 0.002) by the high protein diets. There were no effects (P ≥ 0.309) of CP source on urinary volume or total urinary N, but SBM increased (P ≤ 0.004) urinary urea N, MUN and BUN relative to urea. The proportion of total urinary N that was urea N averaged 75% across diets, and increased (P ≤ 0.003) with high protein diets and with SBM versus urea. This reflects the fact that urea N is the principal form in which excess N is excreted. Because DMI, but not milk protein secretion, was increased by high protein diets, NUE was decreased (P < 0.001) by these diets. Differences observed between urea and SBM-containing diets probably reflect the differences in CP content of these diets. The correlation (r2 ≥ 0.944) of dietary mean MUN with urinary N, urinary urea N, NUE, and ruminal NH3 (Table 5) underscores the close association of MUN with whole-animal N efficiency. Interestingly, MUN was more highly correlated with each of these variables than was BUN. This may be because MUN represents an integrated value of BUN concentrations over the course of the day or else simply the fact that experimental observations for MUN were the mean of six samples, while BUN values were obtained from a single sample. Therefore, although elevated MUN values do not directly indicate the underlying cause, they do appear to be a useful indication of inefficient use of dietary N, in agreement with the observations of others (Broderick and Clayton, 1997). There were no dietary effects (P ≥ 0.133) on total purine derivatives (PD) excretion or the bacterial CP:purine ratio (Table 6). However, each of these responses were numerically decreased by sucrose and numerically increased by high protein diets. Because MPS is a function of the product of these two numbers, MPS was reduced (P = 0.026) by sucrose and increased (P = 0.025) by high protein diets. Chamberlain et al. (1993) observed a significant increase in PD excretion by sheep in response to dietary supplementation with a variety of sugars, but obtained only a small, nonsignificant increase due to wheat starch supplementation. Moreover, a close (r2 = 0.97), negative linear relationship existed between the urinary output of PD and the mean daily ruminal concentration of ammonia, suggesting that MPS was a major sink for the capture of excess ruminally degradable protein (Chamberlain et al., 1993). Khalili and Huhtanen (1991) also showed that the reduction in rumen NH3 by inclusion of sucrose in a grass silage diet was associated with increased MPS. Consistent with these observations, we observed reductions of ruminal NH3 (Table 2 and Figure 1) and MUN concentrations and urinary urea N excretion (Table 4) due Journal of Dairy Science Vol. 85, No. 4, 2002

Figure 2. Residual plots for prediction of urinary N excretion from three alternative equations based on milk urea N. Prediction equations were based on a) Jonker et al., 1998; b) Kauffman and St.Pierre, 1999; or c) R. A. Kohn, 2001. LP = Low protein diet, LP sucrose = low protein sucrose diet, HP urea = high protein urea diet, HP SBM = high protein soybean meal diet.

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to sucrose. However, we also obtained a decrease in MPS due to sucrose. We do not have an explanation for the lack of consistency in our observations; ruminal pH was not adversely affected by sucrose. Microbial protein synthesis was increased 12% by the high protein diets (P = 0.025, CP level) and was increased 8% by SBM relative to urea, but this increase was not significant (P = 0.184, CP source). Amino acids and peptides consistently increase the growth of ruminal bacteria in vitro (Cotta and Russell, 1982; Argyle and Baldwin, 1989; Cruz Soto et al., 1994) when cells are grown on soluble carbohydrates. However, there is less data on the effect of ruminally available amino acids and peptides on microbial growth in vivo. Armentano et al. (1993) reported no response of MPS in midlactation cows to DTP versus NPN. Chikunya et al. (1996) reported increased MPS to DTP in sheep fed sugar beet pulp but not grass hay. These authors inferred that the response of MPS to DTP was a function of the rate of carbohydrate fermentation. Our data regarding the value of DTP relative to NPN are equivocal. We observed a numerical increase in MPS from DTP, but this was perhaps due to an associated increase in ruminal NH3 (Table 2, Figure 1) rather than the provision of free AA and peptides per se. Significant mean bias (P ≤ 0.001) was present for all parameters estimated using the original model of Jonker et al. (1998) and linear bias was present (P ≤ 0.048) for all parameters except N intake (Table 7). In the original model (Jonker et al., 1998), urinary N excretion (g/d) was computed as 12.54 times MUN (mg/ dl); applying this equation in the present study resulted in an underprediction of urinary N by 54.6 g/d. It was subsequently determined that MUN analyses used in the formulation of this model were in error (Kohn et al., 2001). Because each equation in the model relies on predictions from previous equations, errors in the estimation of one parameter are propagated through subsequent predictions. Therefore, two alternative equations relating MUN to urinary N excretion were evaluated, where urinary N excretion is computed as 17.6 times MUN (Kauffman and St.-Pierre, 1999) or urinary N excretion equals 0.026 times BW (in kg) times MUN (R. A. Kohn, 2001, personal communication). A comparison of these alternative equations revealed that mean and linear bias still was present for prediction of urinary N (Figure 2), but the magnitude of the mean bias was reduced substantially (Table 7). Consequently, predictions of subsequent parameters were markedly improved. Mean bias still was significant (P < 0.001) for all remaining predictions, but the magnitude of the bias (less than 15%) was substantially reduced. These results suggest that the original model (Jonker et al., 1998), augmented with an appropriate equation for pre-

dicting urinary N excretion from MUN, is a powerful tool for assessing N efficiency of lactating cows given the simplicity of the inputs required. CONCLUSIONS Replacement of some dietary corn with sucrose decreased ruminal NH3 and MUN concentrations and decreased urinary urea N excretion, all indications of enhanced NUE. However, sucrose also decreased milk yield, milk protein yield, and MPS. A beneficial response to sucrose supplementation may require increased dietary RDP levels to avoid a rumen NH3 limitation. Soybean meal did not significantly increase MPS, DMI, or the yield of milk or milk protein relative to urea. The relative value of NPN versus DTP as sources of RDP for lactating dairy cows remains equivocal. Evaluation of a model that integrated MUN for predicting NUE suggested that MUN can form a simple and noninvasive tool to help assess urinary N excretion from lactating dairy cows. ACKNOWLEDGMENTS The authors would like to thank Kevin Crandall and Pamela Hole for their excellent technical assistance with all aspects of this work. REFERENCES Allison, M. J. 1970. Nitrogen metabolism of ruminal micro-organisms. Pages 456–473 in Physiology of Digestion and Metabolism in the Ruminant. A. T. Phillipson, ed. Oriel Press, Newcastle upon Tyne, UK. Argyle, J. L., and R. L. Baldwin. 1989. Effects of amino acids and peptides on rumen microbial growth yields. J. Dairy Sci. 72:2017–2027. Armentano, L. E., S. J. Bertics, and J. Riesterer. 1993. Lack of response to addition of degradable protein to a low protein diet fed to midlactation dairy cows. J. Dairy Sci. 76:3755–3762. Broderick, G. A., and M. K. Clayton. 1997. A statistical evaluation of animal and nutritional factors influencing concentrations of milk urea nitrogen. J. Dairy Sci. 80:2964–2971. Broderick, G. A., R. J. Wallace, and E. R. Orskov. 1991. Control of rate and extent of protein degradation. Pages 541–592 in Physiological Aspects of Digestion and Metabolism in Ruminants. T. Tsuda, Y. Sasaki, and R. Kawashima, ed. Academic Press, Orlando, FL. Chamberlain, D. G., and J. J. Choung. 1995. The importance of rate of ruminal fermentation of energy sources in diets for dairy cows. Pages 3–27 in Recent Advances in Animal Nutrition. P. C. Garnsworthy and D. J. A. Cole, eds. Nottingham University Press, Nottingham, UK. Chamberlain, D. G., S. Robertson, and J. J. Choung. 1993. Sugars versus starch as supplements to grass silage: Effects on ruminal fermentation and the supply of microbial protein to the small intestine, estimated from the urinary excretion of purine derivatives, in sheep. J. Sci. Food Agric. 63:189–194. Chamberlain, D. G., and P. C. Thomas. 1983. The effect of supplemental methionine and inorganic sulphate on the ruminal digestion of grass silage in sheep. J. Sci. Food Agric. 34:440–446. Chamberlain, D. G., P. C. Thomas, W. Wilson, C. J. Newbold, and J. C. MacDonald. 1985. The effects of carbohydrate supplements Journal of Dairy Science Vol. 85, No. 4, 2002

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