Effect of Dietary Energy and Protein Concentration on the Concentration of Milk Urea Nitrogen in Dairy Ewes1

Effect of Dietary Energy and Protein Concentration on the Concentration of Milk Urea Nitrogen in Dairy Ewes1 A. CANNAS,*,†,2 A. PES,‡ R. MANCUSO,‡ B. VODRET,‡ and A. NUDDA* *Dipartimento di Scienze Zootecniche, Universita’ di Sassari, Sassari 07100, Italy †Department of Animal Science, Cornell University, Ithaca, NY 14853 ‡Istituto Zooprofilattico Sperimentale della Sardegna, Sassari 07100, Italy

ABSTRACT

INTRODUCTION

To study the effects of dietary crude protein ( C P ) and energy on milk urea N concentrations in dairy sheep, eight pelleted total mixed rations were prepared to obtain two levels of energy density (1.65 and 1.55 Mcal of net energy for lactation per kilogram of dry matter for high energy and low energy rations, respectively) and four concentrations of CP within each energy level (mean CP concentrations, 14.0, 16.4, 18.7, and 21.2% of dry matter). The experimental design consisted of two 4 × 4 Latin squares (one per energy level) with two replications per treatment within each 3-wk period. Milk urea N concentrations were similar between dietary energy levels. Within each energy level, milk urea N was linearly and positively associated with dietary CP content and intake (range of milk urea N concentrations, 12.2 to 25.8 mg/dl for ewes fed high energy rations and 12.9 to 26.7 mg/dl for ewes fed low energy rations). The comparison of these results with those from other trials suggested that milk and blood urea N concentrations are closely correlated with dietary CP concentrations and less closely correlated with dietary CP intake. Our results suggest that milk or blood urea N concentrations can be used as indicators of protein metabolism and intake of lactating ewes. ( Key words: milk urea, dietary energy, dietary protein, sheep)

The milk production of sheep is an important economic activity in many countries. Ovine milk is processed to produce cheese, yogurt, and other dairy products. In 1993, about 21 million ewes in Mediterranean countries of the European Union produced a total of 1985 × 106 L of ovine milk, and the world production of ovine milk was about 8091 × 106 L (15). Accurate prediction of pasture quality and intake is economically important because most dairy sheep are raised on pasture. Without this information, the development of appropriate supplementation strategies to provide a balanced ration is difficult. Nutritional indicators could be useful to assess the adequacy of the diet. Blood urea N ( BUN) concentrations and milk urea N ( MUN) concentrations are considered to be good indicators of protein metabolism and intake in dairy cows (29, 32) and are currently used as tools to evaluate diets. Milk urea N and BUN are closely associated (21, 29), although MUN tends to be a more stable parameter ( 4 ) and is more easily sampled. Milk urea is a noncasein N component of milk (2.5 to 3.0% of the total milk N in cows) that does not contribute to cheese production. High BUN and MUN have been associated with low reproductive efficiency in cows (8, 14). The relationship between N intake and utilization has been extensively studied in growing lambs and in dry ewes. Tagari et al. ( 3 5 ) found that BUN and N retention were significantly and positively correlated and that urea was a good index of protein utilization. Blood urea was positively and linearly related to the urea entry rate (quantity of urea synthesized or liberated into the body pool per unit of time) ( 1 2 ) and resulted in a very effective basis for the prediction of N utilization (13). However, in other cases, BUN increased as N intake increased up to a certain level and then reached a plateau. Above that point, further increases in N intake did not cause further increases in BUN (25). Moreover, in a review on this subject,

Abbreviation key: BUN = blood urea N, HE = high energy, LE = low energy, MUN = milk urea N, NSC = nonstructural carbohydrate.

Received February 26, 1997. Accepted September 24, 1997. 1This study was supported by the Ministero dell’Universita’ e della Ricerca Scientifica e Tecnologica, Italy (Grant type: 60%). 2Author to whom correspondence should be addressed. 1998 J Dairy Sci 81:499–508

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Harmeyer and Martens ( 1 9 ) concluded that changes in BUN do not always reflect changes in the total entry rate of urea and may occasionally lead to erroneous conclusions when used for its prediction. Another important aspect of urea metabolism in ewes is that high BUN (between 15.8 and 23.2 mg/dl) was associated with detrimental effects on early development and survival of sheep embryos ( 7 ) . Concentrations of BUN or MUN could be good indicators of N intake and utilization in lactating ewes as well. However, the information obtained with BUN in dry ewes is of limited value because lactating ewes have much higher nutrient requirements and intakes. Lactating ewes also require diets that are much richer in energy and protein. Therefore, the definition of MUN or BUN reference values for lactating ewes would be beneficial. These values could serve as indicators of protein metabolism and utilization and could help to improve the reproductive efficiency of sheep. This study was designed to evaluate the relationships between dietary CP and MUN in lactating dairy ewes fed rations with a wide range of CP concentrations and with two energy levels. MATERIALS AND METHODS Trial Sixteen Sarda ewes that were more than 90 DIM and had a mean BW of 43 kg were kept in individual pens and were fed pelleted TMR during this trial. Eight different pelleted TMR were prepared (Table 1 ) to obtain two levels of energy density and four concentrations of CP within each energy level. High energy ( HE) TMR had a calculated NEL concentration of 1.65 Mcal/kg of DM and CP concentrations of 14.2, 16.6, 18.8, and 21.2% (DM basis). Low energy ( LE) TMR had a calculated NEL concentration of 1.55 Mcal/kg of DM and CP concentrations of 13.9, 16.3, 18.6, and 21.1% (DM basis). The energy content of the feeds was calculated as the weighted mean of the energy concentration ( 2 6 ) of the ingredients. For each ingredient, TDN at the maintenance level was estimated on the basis of the TDN content of feedstuffs with a similar chemical composition as that described by the NRC ( 2 6 ) and Van Soest (36). During the 12-d preliminary period, all ewes received a homogeneous mixture of all eight TMR. At the end of this period, ewes were divided into two groups of 8 ewes each based on production (group 1 produced 1.28 kg/d of milk, and group 2 produced 1.22 kg/d; SEM = 0.05 kg/d). During the experimental Journal of Dairy Science Vol. 81, No. 2, 1998

period ewes in group 1 were fed only the HE TMR, and ewes in group 2 received only LE TMR, following an experimental design with two (HE and LE) 4 × 4 Latin squares with two replications per treatment within each 3-wk period ( 8 ewes per Latin square). A treatment sequence in which treatment is preceded equally often in the design by each of the other treatments that was developed by Williams and presented by Cochran and Cox ( 1 1 ) was used to estimate residual effects. Measurements During the last 3 d of each experimental period, individual daily feed intake and daily milk production were measured. Individual milk samples were taken at each of the two daily manual milkings (12-h interval). Chemical Analyses Feed samples were analyzed for NDF after urea ( 8 M) treatment and without sodium sulfite (38), ADF and acid detergent lignin (16), fat ( 2 ) , ash, CP ( 3 ) , and CP fractions (22, 34). The nonstructural carbohydrate ( NSC) concentration of the rations was calculated as 100 – (NDF – NDIP) – CP - ether extract – ash, where NDIP = neutral detergent insoluble protein. Milk analyses were performed on each sample from each milking, and a 3-d weighted mean was calculated for each ewe and each experimental period. Milk fat content was estimated using the Roese-Gottlieb method ( 1 ) . The true protein content of milk was estimated by direct determination using Kjeldahl analysis on the precipitate after treatment with trichloroacetic acid ( 6 ) . Lactose was determined by an enzymatic method (kit number 986–119; BoehringerMannheim, Mannheim, Germany). Milk pH was measured using a pH meter with a temperature probe (Expandable Ion Analyzer model EA940 and electrode Ross model 81–02; Research Inc., Boston, MA). The MUN was determined by an enzymatic (urease and glutamate dehydrogenase) colorimetric method (kit number 542–946; Boehringer-Mannheim) after milk was treated with trichloroacetic acid and centrifuged. Spectrophotometer absorbance was set at 340 nm. Statistical Analyses Each of the two Latin squares was analyzed separately; residual treatment effects were estimated separately also (11). For reasons unrelated to treat-

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ment, the data of one ewe in the LE TMR Latin square were omitted for one period. The statistical analyses, therefore, were carried out after estimation of the missing values (11). Data were analyzed using the following model: Yijk = m + ai + bj + tk + rk + eijk where m = overall mean, ai = main effect of the blocking variable (animal), bj = main effect of the blocking variable (period),

tk = direct main effect of treatment, rk = residual effect depending on the treatment applied during the preceding period, and eijk = random error. When treatment effect was significant, the sum of squares was partitioned into sums of squares for quantitative (linear, quadratic, and cubic) contrasts (27). After checking the equality of variance with the Bartlett test (27), the overall means of the two Latin squares were compared using a two-tailed t test. Morning and evening MUN were also compared using a t test. Dietary CP concentration and intake were

TABLE 1. Composition of the rations fed to ewes. HE1 Composition

14.2% CP3

16.6% CP

LE2 18.8% CP

21.2% CP

13.9% CP

16.3% CP

18.6% CP

21.1% CP

( % of DM) Ingredient Dehydrated alfalfa 30.22 Beet pulp 13.15 Soybean hulls 14.63 Ground corn grain 19.71 Wheat grain 20.39 Fish meal . . . Soybean meal . . . Corn gluten meal . . . Calcium carbonate 0.19 Calcium phosphate 0.83 Mineral and vitamin mix4 0.44 Lignosulfate 0.44 Chemical CP 14.2 2.5 EE5 Ash 7.6 NDF 38.1 ADF 22.7 Acid detergent lignin 3.4 41.8 NSC6 NSC:CP 2.9 Protein fractions7 A 2.0 1.0 B1 7.2 B2 3.2 B3 C 0.9

30.21 14.11 14.79 18.02 15.32 0.69 4.45 0.68 0.16 0.69 0.44 0.44

30.20 15.01 14.94 16.44 10.55 1.34 8.62 1.33 0.14 0.55 0.44 0.44

30.18 15.98 15.10 14.76 5.49 2.03 13.07 2.01 0.10 0.40 0.44 0.44

29.97 38.98 24.98 3.91 . . . . . . . . . . . . 0.08 1.20 0.44 0.44

33.39 32.30 24.99 2.58 . . . 0.69 3.43 0.68 0.08 0.98 0.44 0.44

36.60 26.01 25.01 1.33 . . . 1.34 6.65 1.32 0.08 0.78 0.44 0.44

40.03 19.33 25.02 . . . . . . 2.02 10.08 2.00 0.08 0.56 0.44 0.44

16.6 2.3 7.5 38.0 22.8 3.5 40.3 2.4

18.8 2.2 7.4 37.9 23.0 3.6 38.9 2.1

21.2 2.0 7.3 37.7 23.1 3.6 37.4 1.7

13.9 1.9 8.2 49.4 32.0 4.2 32.5 2.3

16.3 2.2 8.6 48.7 32.2 4.3 29.9 1.8

18.6 2.5 9.0 48.0 32.4 4.4 27.4 1.5

21.1 2.9 9.4 47.3 32.5 4.6 24.8 1.2

2.2 0.9 8.9 3.5 1.0

2.3 0.8 10.7 3.9 1.1

2.4 0.6 12.8 4.2 1.3

1.8 0.5 6.1 4.4 1.1

2.1 0.8 7.9 4.4 1.2

2.3 1.0 9.8 4.3 1.3

2.4 1.4 12.0 3.9 1.3

1High

energy (1.65 Mcal of NEL/kg of DM). energy (1.55 Mcal of NEL/kg of DM). 3All CP concentrations are on a DM basis. 4Guaranteed analysis: 20% magnesium carbonate, 150 ppm of Co, 20,000 ppm of Fe, 500 ppm of I, 12,000 ppm of Mn, 20 ppm of Se, 30,000 ppm of Zn, 4,000,000 IU/kg of vitamin A, 800,000 IU/kg of vitamin D3, and 4000 ppm of vitamin E acetate. 5Ether extract. 6Nonstructural carbohydrates, calculated as 100 – (NDF – NDIP) – CP – EE – ash, where NDIP = neutral detergent insoluble protein. 7Protein fractions: A = NPN, B = buffer-soluble true protein, B = buffer-insoluble protein – neutral detergent soluble protein, B = 1 2 3 neutral detergent insoluble protein – acid detergent insoluble protein, and C = acid detergent insoluble protein (22, 34). 2Low

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TABLE 2. Nutrient intake and milk production and composition by ewes fed diets with 1.65 Mcal of NEL/kg of DM. Dietary CP content (DM basis) Treatment effect

14.2% CP

16.6% CP

18.8% CP

21.2% CP

SEM1

Direct

Residual

DMI, kg/d 2.03 Energy intake, Mcal/d 3.35 CP Intake, g/d 288 Milk production, kg/d 1.16 Fat, % 5.7 Fat yield, g/d 63.9 True protein, % 5.7 True protein yield, g/d 64.2 Lactose, % 4.6 pH 6.62 Urea N, mg/dl 12.2 Urea N, % of true protein N 1.4

2.34 3.87 389 1.20 5.7 67.7 5.4 65.0 4.5 6.59 17.0 2.0

2.33 3.84 438 1.34 5.4 70.9 5.3 69.6 4.5 6.60 22.3 2.4

2.12 3.49 449 1.34 5.6 73.2 5.4 70.0 4.6 6.59 25.8 3.1

0.17 0.40 27 0.07 0.1 3.4 0.1 4.0 0.1 0.03 1.1 0.2

NS2 NS 0.01 0.13 0.21 NS 0.05 NS NS NS 0.001 0.001

NS NS NS NS 0.19 0.22 NS NS 0.25 0.11 NS NS

Components Linear

Quadratic

NS NS 0.001 NS NS NS 0.03 NS NS NS 0.001 0.001

NS NS 0.10 NS NS NS 0.14 NS NS NS NS NS

P

1n 2P

= 8. > 0.2.

regressed against MUN by simple linear regression. When necessary, the equality of two regression equations was tested using a method based on indicator variables (27). RESULTS The chemical compositions of the eight pelleted TMR are reported in Table 1. The CP concentrations were similar between the HE and LE TMR. The HE TMR had lower NDF and higher NSC concentrations than did the LE TMR. The CP fractions were fairly similar between the HE and LE TMR; there was a large prevalence of the B2 fraction (buffer insoluble protein minus the protein soluble in neutral detergent). HE Latin Square Dry matter and calculated NEL intakes were not affected by treatment (Table 2). However, an increase in dietary CP concentrations resulted in quadratic ( P < 0.1) increases in CP intake. Milk production tended to increase as the CP concentration in the ration increased ( P < 0.13). Milk fat content and yield, milk protein yield, milk lactose concentration, and milk pH were similar for ewes fed all dietary CP concentrations. Milk CP concentrations were highest for ewes fed the TMR with the lowest CP concentration. Milk urea N concentration and MUN as a percentage of the true protein N content of the milk increased linearly ( P < 0.001) as the CP concentration of the TMR increased. Residual effects and cubic contrasts were not significant for any of these parameters. Milk urea N concentrations did not differ Journal of Dairy Science Vol. 81, No. 2, 1998

between morning and evening milkings (morning, 19.3 mg/dl; evening, 19.5 mg/dl; SEM = 1.2). LE Latin Square Dry matter and calculated NEL intakes were not affected by dietary CP content but tended to be lower for ewes fed TMR with CP concentrations of 13.9 and 16.3% (Table 3). An increase in dietary CP concentrations resulted in linear ( P < 0.001) increases in CP intake, MUN concentration, and MUN as a percentage of the true protein N content of the milk. Increased dietary CP concentration resulted in a quadratic increase in milk production ( P < 0.07), milk fat yield ( P < 0.07), and milk true protein yield ( P < 0.02). Cubic contrasts were significant only for milk pH ( P < 0.03). Milk fat, protein, and lactose contents were not significantly different among treatments. Residual effects were not significant for any of the parameters studied except for milk pH ( P < 0.04), MUN concentration ( P < 0.08), and MUN as a percentage of the true protein N content of the milk ( P < 0.1). However, for these milk components, the residual effects were always very small. Therefore, these residual effects were considered to be of little biological interest and were not added to the treatment means in Table 3. The MUN concentrations did not differ between morning and evening milkings (morning, 20.3 mg/dl; evening, 19.4 mg/dl; SEM = 1.1). HE Latin Square Versus LE Latin Square Although ewes ate more of the LE TMR than of the HE TMR ( P < 0.06), the calculated daily NEL intake

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MILK UREA IN DAIRY EWES TABLE 3. Nutrient intake and milk production and composition by ewes fed diets with 1.55 Mcal of NEL/kg of DM. Dietary CP content (DM basis) Treatment effect

13.9% CP

16.3% CP

18.6% CP

21.1% CP

SEM1

Direct

Residual

DMI, kg/d 2.18 Energy intake, Mcal/d 3.37 CP Intake, g/d 302 Milk production, kg/d 1.26 Fat, % 6.0 Fat yield, g/d 73.8 True protein, % 5.5 True protein yield, g/d 67.4 Lactose, % 4.6 6.59 pH3 Urea N, mg/dl 12.9 Urea N, % of true protein N 1.5

2.38 3.69 389 1.43 5.7 81.1 5.4 76.9 4.7 6.62 17.7 2.1

2.54 3.94 473 1.50 5.7 83.7 5.3 77.1 4.6 6.67 23.4 3.0

2.47 3.83 529 1.48 5.9 85.2 5.2 75.5 4.6 6.61 26.7 3.3

0.10 0.16 17 0.05 0.1 1.5 0.1 2.3 0.1 0.02 0.6 0.1

0.15 0.15 0.001 0.01 NS 0.001 0.16 0.01 NS 0.01 0.001 0.001

NS2 NS NS NS NS 0.15 NS NS NS 0.04 0.08 0.10

Components Linear

Quadratic

NS NS 0.001 0.004 NS 0.001 NS 0.03 NS 0.07 0.001 0.001

NS NS NS 0.07 NS 0.07 NS 0.02 NS 0.008 NS 0.13

P

1n

= 8. > 0.2. 3Milk pH also had a significant cubic component of the variance ( P < 0.03). 2P

was not affected by dietary NEL concentration (Table 4). Daily CP intake, milk production, milk fat content and yield, milk true protein yield, milk lactose content, and MUN as a percentage of the true protein N content of milk were higher for ewes fed the LE TMR. Milk pH and concentrations of milk protein and MUN concentration were not affected by treatment. Relationships Between MUN and CP Content of the Rations When the MUN concentration of each ewe was regressed against the CP concentration of the rations, the coefficient of determination was lower for the HE TMR than that for the LE TMR (0.62 vs. 0.70) (Table 5, Equations [1] and [2]). The regression equations were almost identical and were not statistically different from the regression based on the pooled HE and LE data (Table 5, Equation [3]; Figure 1A). When the MUN concentration of each ewe was plotted against the daily intake of CP, a similar trend was observed but with lower coefficients of determination (HE TMR, 0.37; LE TMR, 0.50) (Table 5, Equations [4] and [5]). In this case, the regression equations were almost identical and could be pooled (Table 5, Equation [6]; Figure 1B). If the total amount of MUN (milligrams per day) was the dependent variable instead of the concentration of MUN (milligrams per deciliter), the coefficient of determination was dramatically decreased for each of the independent variables considered. For this reason, the amount of MUN is not reported as a variable.

As would be expected, when treatment means, instead of individual ewe data, were used, the relationships between MUN and dietary CP content or intake were much closer (Table 5, Equations [7], [8], [9], [10], [11], and [12]; Figures 2 and 3). For both CP concentration and CP intake, the regression equations calculated separately for the HE and LE TMR were not significantly different from the regression based on the pooled HE and LE data. Dietary CP concentration was slightly more closely related with MUN than was the daily intake of CP (Table 5, Equations [9] and [12]).

TABLE 4. Effect of high energy ( H E ) or low energy ( L E ) concentration on nutrient intake and milk production and composition. Energy level HE1 DMI, kg/d 2.20 Energy intake, Mcal/d 3.64 CP Intake, g/d 391 Milk production, g/d 1.26 Fat, % 5.58 Fat yield, g/d 68.9 True protein, % 5.45 True protein yield, g/d 67.2 Lactose, % 4.55 pH 6.60 Urea N, mg/dl 19.3 Urea N, % of protein N 2.2

LE2

SEM3

P

2.39 3.71 424 1.42 5.82 81.1 5.36 74.4 4.63 6.62 19.9 2.4

0.07 0.09 11 0.03 0.06 1.25 0.07 1.5 0.03 0.01 0.64 0.1

0.06 NS4 0.04 0.001 0.007 0.001 NS 0.003 0.07 NS NS 0.04

11.65

Mcal of NEL/kg of DM. Mcal of NEL/kg of DM. 3n = 32. 4P > 0.2. 21.55

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Figure 2. Relationship between treatment means for milk urea N concentrations of ewes fed high energy ( H E ) TMR ( —◊––; 1.65 Mcal of NEL/kg of DM) and those of ewes fed low energy ( L E ) TMR ( - - -⁄- - -; 1.55 Mcal of NEL/kg of DM) and dietary CP content. Regression equations (numbers in parentheses are standard errors of the coefficients): y = 1.97 (0.12)x – 15.58 (2.22), r2 = 0.99 (HE TMR) and y = 1.97 (0.16)x – 14.35 (2.74), r2 = 0.99 (LE TMR).

MUN was negatively associated with the dietary concentration of NSC ( r = –0.35; P < 0.01). However, the range of variation of NSC and NDF within squares was much smaller than that of CP, reducing the importance of correlation analysis for them. Figure 1. Relationship between the concentration of milk urea N for individual ewes fed high energy TMR ( ◊; 1.65 Mcal of NEL/kg of DM) or low energy TMR ( ⁄; 1.55 Mcal of NEL/kg of DM) and dietary CP content ( A ) or dietary intake of CP ( B ) . Regression equations pooled over energy treatments (numbers in parentheses are standard errors of the coefficients): y = 1.96 (0.18)x – 14.78 (3.26), r2 = 0.65 ( A ) and y = 0.04 (0.006)x + 2.74 (2.55), r2 = 0.43 (B).

The correlation analysis between pooled (HE and LE) individual data and daily intake of each dietary CP fraction showed a positive association between MUN concentration and intake of each CP fraction; the highest association was observed for MUN and the B2 fraction [fraction A (NPN), r = 0.54, P < 0.01; B1 (buffer- soluble true protein), r = 0.34, P < 0.01; fraction B2 (buffer-insoluble protein – neutral detergent soluble protein), r = 0.74, P < 0.01; fraction B3 (neutral detergent insoluble protein – acid detergent insoluble protein), r = 0.32, P < 0.05; and fraction C (acid detergent insoluble protein), r = 0.52, P < 0.01. The MUN did not appear to be associated with NSC intake ( r = –0.05), NDF intake ( r = –0.01), or the dietary concentration of NDF ( r = –0.02), although Journal of Dairy Science Vol. 81, No. 2, 1998

DISCUSSION Intake Although DMI was higher for ewes fed LE TMR than for those fed HE TMR for all CP percentages, this difference was more pronounced at the highest CP concentration. Intake of NEL was similar between ewes fed the HE and LE TMR, which suggests that intake was regulated by energy demand. The pelleted TMR were finely ground and unlikely to impose physical restrictions on intake (10). Milk Production and Composition Milk production tended to increase as the dietary CP concentration increased up to the third highest dietary CP concentration in both Latin squares. Above this percentage, (ca. 18.7%), the production of milk tended to reach a plateau. However, this curvilinear trend was significant only in the LE TMR Latin square ( P < 0.07). Dietary CP concentrations that maximized milk production were higher than those suggested by some feeding systems for sheep

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TABLE 5. Linear regressions describing the relationships between concentrations of milk urea N (MUN) and either dietary CP content or intake of CP for high energy ( H E ) and low energy ( L E ) rations. Regression coefficients

Equation

Y

X

Energy level

Intercept

[1] [2] [3] [4] [5] [6]

MUN2 MUN MUN MUN MUN MUN

CP3 CP3 CP3 CP4 CP4 CP4

HE LE Both HE LE Both

–15.58 –14.35 –14.78 2.62 2.49 2.74

[7] [8] [9] [10] [11] [12]

MUN MUN MUN MUN MUN MUN

CP3 CP3 CP3 CP4 CP4 CP4

HE LE Both HE LE Both

–15.58 –14.35 –14.78 –10.95 –5.96 –6.93

SE1

Slope

Data for individual ewes 5.07 1.97 4.15 1.97 3.26 1.96 4.09 0.04 3.33 0.04 2.55 0.04 Treatment means 2.22 1.97 2.74 1.97 2.29 1.96 6.76 0.08 0.76 0.06 3.05 0.07

SE1

SE of Estimates r2

P

n

0.28 0.23 0.18 0.01 0.008 0.006

4.19 3.54 3.87 5.37 4.60 4.92

0.62 0.70 0.65 0.37 0.50 0.43

0.001 0.001 0.001 0.001 0.001 0.001

32 32 64 32 32 64

0.12 0.16 0.13 0.02 0.002 0.007

0.65 0.83 0.96 2.17 0.30 1.61

0.99 0.99 0.97 0.95 1.00 0.93

0.01 0.006 0.001 0.03 0.001 0.001

4 4 8 4 4 8

1Standard

errors of the regression coefficients. in milligrams per deciliter. 3Measured as a percentage of total DM. 4Measured in grams per day. 2Measured

( 2 0 ) but were similar to those that maximized milk production in lactating ewes fed fish meal as the main protein source (18, 31). The pelleted TMR used in our trial were finely ground. Their high intake might have affected the CP fermentation pattern (with high escape of proteins) compared with that usually assumed to occur in most sheep feeding systems, which are based on data from nonlactating ewes fed more conventional diets (37). Milk production was lower at each dietary CP concentration in the HE TMR Latin square than in the LE TMR Latin square, although at the beginning of the experiment ewes fed the HE TMR were slightly more productive. Furthermore, the HE TMR provided higher NSC concentrations and lower NDF concentrations than did the LE TMR. Two compatible explanations are possible. First, the high NSC concentration of the HE TMR could have induced subclinical acidosis in the ewes and thus impaired milk production. Indeed, milk fat content was lower ( P < 0.007) for ewes fed the HE TMR, despite their lower milk production, suggesting an episode of milk fat depression caused by an excess of NSC (36). Second, the large amount of NSC in the HE TMR might have stimulated propionate production and subsequent insulin production. In ewes in the second part of lactation, this effect would have stimulated nutrient uptake by tissues, resulting in decreased lipolysis, milk production, and milk fat yield and increased body fat deposition, following a mechanism suggested by Ørskov (30). However, in this trial we did not measure

BW variations, and, thus, we cannot support or disprove this hypothesis. MUN In both Latin squares, MUN concentration increased linearly (Tables 2 and 3 ) as CP concentration and intake increased. This result appears to be in

Figure 3. Relationship between treatment means for milk urea N concentrations of ewes fed high energy ( H E ) TMR (—◊––; 1.65 Mcal of NEL/kg of DM) and those of ewes fed low energy ( L E ) TMR ( - - -⁄- - -; 1.55 Mcal of NEL/kg of DM) and dietary CP intake. Regression equations (numbers in parentheses are standard errors of the coefficients): y = 0.08 (0.02)x – 10.95 (6.76), r2 = 0.95 (HE TMR) and y = 0.06 (0.002)x – 5.96 (0.76), r2 = 1.00 (LE TMR). Journal of Dairy Science Vol. 81, No. 2, 1998

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contrast to the findings of McIntyre (25), who reported that, in dry ewes, BUN increased up to 30 mg/dl as CP intake increased up to about 150 g/d. Above that point, further increases in CP intake did not cause further increases in BUN. However, we have found that even in his data (25), the relationship between BUN and dietary CP concentration was statistically linear for the whole range of data. Despite LE TMR had lower NEL and NSC concentrations and the higher daily CP intakes of ewes fed those rations compared with the HE TMR, no differences in MUN were found. This result was unexpected, because the higher dietary NSC content of the HE TMR should have provided more fermentable energy in the rumen, which should reduce ruminal ammonia concentrations, resulting in lower urea production by the liver. Indeed, in dairy cows, the ratio between dietary protein and energy was more related to MUN than was the daily intake of CP (29). We have proposed one mechanism that may explain the similar MUN concentrations between energy levels. Because of the high intake observed in this trial, the passage rate of the feeds was probably high. If so, a large amount of protein might have escaped from the rumen and reached the intestine. The concentration of MUN would then be more related to the total amount of protein absorbed from the intestine than to the amount of protein fermented in the rumen. Indeed, even in nonlactating ewes, the gluconeogenesis from amino acids was found to be more important than rumen ammonia concentration as a source of blood urea variation ( 5 ) . Another effect of low rumen protein fermentation would be that low amounts of NSC would be necessary to maximize the utilization of rumen ammonia by bacteria. These facts could explain the very high coefficient of determination between CP and MUN in our regressions and the very small differences between energy levels. When MUN was regressed against dietary CP concentration or the daily intake of CP using all individual ewe data, the coefficient of determination was lower for ewes fed the HE TMR than for those fed the LE TMR, and was higher when dietary CP concentration was used as regressor instead of the daily intake of CP (Table 5, Equations [1], [2], [4], and [5]). When only the mean of each treatment was used, the coefficient of determination was greatly increased (more than 0.9) for both Latin squares and for both independent variables (Table 5, Equations [7], [8], [9], [10], [11], and [12]). Large individual variation in MUN concentrations among ewes fed the same rations occurred. This amount of variation suggests that interpretation of MUN data should be based on Journal of Dairy Science Vol. 81, No. 2, 1998

means rather than on individual observations. The regression analysis showed a very close relationship between MUN and either dietary CP content or daily intake of CP (Table 5, Equations [7], [8], [9], [10], [11], and [12]). Because the differences in feed intake were small within each energy level, it is difficult to conclude from our data whether dietary CP concentration or daily intake of CP is a better predictor of MUN. To determine whether the close relationships between MUN and dietary CP of our trial would exist under other conditions, we compared our data with those available in the literature. No other trials have specifically studied the relationship between MUN or BUN and dietary CP in lactating ewes. However, data on BUN in lactating ewes are available from trials in which the main goal was to study the effect of dietary protein on milk production (9, 17, 18, 23, 24, 28, 33). In those studies, 61 dietary treatments with seven different protein supplements were considered. All diets were fed as TMR. The relationship between BUN (data from the previously mentioned trials) and MUN (data from our trial) and either dietary CP content or daily intake of CP is shown in Figure 4 ( A and B). A close and linear relationship between dietary CP concentration and urea N (Figure 4A) was observed. The MUN data from our experiment fit very well with the BUN data obtained in the other experiments. The coefficient of determination was surprisingly high (0.82), considering the variety of situations present in those experiments (different diets, breeds, and stages of lactation). Moreover, this regression equation (Figure 4A) was similar to the one found in our experiment (Table 5, Equation [9]). When the same data were used to relate MUN or BUN and daily protein intake, we obtained a regression equation (Figure 4B) that was similar to the corresponding one found in our experiment (Table 5, Equation [12]). However, the coefficient of determination was much lower than that of the regression in Figure 4A in which dietary CP concentration was used as an independent variable ( r 2 = 0.56 vs. 0.82). Similarly, in dry sheep, rumen ammonia was found to be more closely correlated with dietary CP content than with daily intake of CP (13). These facts suggest that the ratios among nutrients in the diet are more important than the daily intake of nutrients in controlling blood urea concentration. If the close relationship of MUN and BUN with dietary CP content exists for grazing animals as well, the measurement of MUN could be used to estimate the CP content of the diet consumed by this type of animal. This approach appears to be promising, and

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energy levels considered. The differences in MUN between energy levels were very small and nonsignificant. Comparison of the results of our experiment with other trials suggested that MUN and BUN are closely correlated with dietary CP concentration. Thus, MUN may be used to estimate the CP content of the diet fed to ewes or, with lower precision, their CP intake, which would be especially important for grazing animals for which intake estimation represents a major problem. Additional experiments are required to study the relationships among dietary CP, CP requirements, and MUN with more conventional diets and with grazing animals. The study of the relationship between MUN and reproductive efficiency of ewes should also be considered in the definition of MUN reference values, which could increase the importance of the utilization of this nutritional indicator in the field. ACKNOWLEDGMENTS The authors gratefully thank Alice Pell and Peter J. Van Soest for the helpful suggestions. The technical assistance provided by Roberto Rubattu of the University of Sassari and by the technicians of the Chimica Bromatologica laboratory of the Istituto Zooprofilattico Sperimentale della Sardegna of Sassari is appreciated. REFERENCES

Figure 4. Relationships between CP content of the TMR ( A ) or daily CP intake ( B ) and concentration of milk or blood urea in housed lactating ewes. Data for milk urea N ( ◊) are from this trial, and data for blood urea N ( ⁄) are from previously published papers (9, 17, 18, 23, 24, 28, 33). Each square represents a treatment mean. Regression equations (numbers in parentheses are standard errors of the coefficients): y = 2.09 (0.12)x – 18.14 (1.78), r2 = 0.82 ( A ) and y = 0.06 (0.005)x – 8.35 (2.35), r2 = 0.56 ( B ) .

similar experiments carried out specifically on grazing animals should be conducted. At the CP concentration at which milk production was maximized, MUN was potentially high enough to cause negative effects on the reproductive efficiency of the ewes ( 7 ) . CONCLUSIONS In this experiment, the concentration of MUN was linearly related to the CP content of the diets for both

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