Available online at www.sciencedirect.

com

Animal Feed Science and Technology

140 (2008) 191–198

Short communication
Effects of dietary urea levels on milk
protein fractions of Holstein cows
A.A. Aquino a , Y.V.R. Lima a , B.G. Botaro a , C.S.S. Alberto a ,
K.C. Peixoto Jr. a , M.V. Santos b,∗
a Department of Animal Nutrition and Production, School of Veterinary Medicine and Animal Science,
University of São Paulo, Pirassununga, Brazil
b Universidade Metodista de São Paulo, São Bernardo do Campo, SP, Brazil

Received 21 August 2006; received in revised form 1 March 2007; accepted 8 March 2007

Abstract

The aim of this study was to evaluate the effects of substituting soybean meal for urea on milk
protein fractions (casein, whey protein and non-protein nitrogen) of dairy cows in three dietary lev-
els. Nine mid-lactation Holstein cows were used in a 3 × 3 Latin square arrangement, composed of
3 treatments, 3 periods of 21 days each, and 3 squares. The treatments consisted of three different
diets fed to lactating cows, which were randomly assigned to three groups of three animals: (A) no
urea inclusion, providing 100% of crude protein (CP), rumen undegradable protein (RUP) and rumen
degradable protein (RDP) requirements, using soybean meal and sugarcane as roughage; (B) urea
inclusion at 7.5 g/kg DM in partial substitution of soybean meal CP equivalent; (C) urea inclusion
at 15 g/kg DM in partial substitution of soybean meal CP equivalent. Rations were isoenergetic and
isonitrogenous—160 g/kg DM of crude protein and 6.40 MJ/kg DM of net energy for lactation. When
the data were analyzed by simple polynomial regression, no differences were observed among treat-
ments in relation to milk CP content, true protein, casein, whey protein, non-casein and non-protein
nitrogen, or urea. The milk true protein:crude protein and casein:true protein ratios were not influ-
enced by substituting soybean meal for urea in the diet. Based on the results it can be concluded that
the addition of urea up to 15 g/kg of diet dry matter in substitution of soybean meal did not alter milk

Abbreviations: CP, crude protein; NPN, non-nitrogen protein; RUP, rumen undegradable protein; RDP, rumen-
degradable protein; DM, dry matter
∗ Corresponding author at: Av. Duque de Caxias Norte, 225, Campus USP, Pirassununga 13635-900, SP, Brazil.

Tel.: +55 19 35654240; fax: +55 19 35654227.

E-mail address: mveiga@usp.br (M.V. Santos).

0377-8401/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2007.03.005
192 A.A. Aquino et al. / Animal Feed Science and Technology 140 (2008) 191–198

protein concentration casein, whey protein and its non-protein fractions, when fed to lactating dairy
cows.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Dairy cow; Urea; Non-protein nitrogen; Milk urea nitrogen; Casein; Whey protein

1. Introduction

The interest in milk protein has risen in the last decades due to its nutritional (Meisel,
2004) and economical importance (Emmons et al., 2003). Higher true protein concentration
in milk is desirable to attend the dairy industry demands (Emmons et al., 2003). Milk
nitrogen fractions are composed of casein, whey proteins and non-protein nitrogen (Depeters
and Cant, 1992). Casein and whey protein constitute the milk true protein fraction (Farrell
et al., 2004). The non-protein nitrogen (NPN) fraction often corresponds to 50–60 g/kg of
milk total nitrogen, and almost 50% of this NPN fraction is constituted of urea (Depeters
and Cant, 1992).
Efficient rumen microbial protein synthesis is dependent on synchronization of optimal
energy and adequate ratio of metabolizable energy:rumen degradable nitrogen, allowing
microorganisms to capture N into microbial protein (Hoover and Stokes, 1991). Sugarcane
and non-protein nitrogen sources, like urea, are widely used to feed dairy cows due to their
low cost, high yield capacity per hectare, and because sugarcane has its production spread
along the year.
Sugarcane may be fed to dairy cows, however, its main nutritional limitation is the
indigestibility of the fiber fraction, leading to reduced dry matter intake (DMI). Other
nutritional limitations include low lipids, low minerals and protein concentration (specially
limited sulfured amino acids), low starch content, and high levels of readily fermentable
carbohydrates (Magalhães et al., 2004). Mendonça et al. (2004) reported that the use of
sugarcane decreases DMI, increases concentrate DMI and decreases milk yield, but no
difference was found on milk composition.
Bovine milk protein concentration can be altered by several nutritional factors, e.g., dry
matter intake, energy and protein ratio, quality and digestibility of fiber, dimensions and
density of feed particles, feeding frequency, and nitrogen sources (Santos and Huber, 1996).
Several researches have been conducted to determine the effects of different nitrogen sources
on concentration and milk protein yield when fed to lactating cows (Cameron et al., 1991;
Oliveira et al., 2001; Silva et al., 2001). However, only few studies have been published con-
cerning the effects of urea inclusion in diets of lactating dairy cows on milk protein fractions,
i.e. casein, whey protein and non-protein nitrogen (Roseler et al., 1993; Baker et al., 1995).
We hypothesized that the substitution of soybean meal by urea as NPN source in a
sugarcane based diet fed to lactating dairy cows may alter milk protein synthesis and milk
protein fractions. Therefore, the objective of this study was to determine the effects of
substituting soybean meal for three levels of urea equivalent (0, 7.5 or 15 g/kg DM) on
the concentration of total milk protein, casein, whey protein and non-protein nitrogen in
Holstein cows.
 A.A. Aquino et al. / Animal Feed Science and Technology 140 (2008) 191–198 193

2. Materials and methods

2.1. Experimental design

Nine mid-lactation Holstein cows, averaging 560 kg of body weight and with milk
somatic cell counts bellow 300,000 cells/ml, were selected for this study. Cows were
arranged in a 3 × 3 Latin square design, composed of 3 treatments, three 21-day peri-
ods (total of 63 days) and 3 squares. In each of the 21-day period, cows had the first 17
days for diet adaptation, and the last 4 days of the period for milk samples collection used
for analysis. All cows were milked twice a day.

2.2. Experimental diets

Cows were randomly assigned to receive one of the three diets (Table 1): (A) no urea
inclusion, providing 100% of crude protein (CP), rumen undegradable protein (RUP) and
rumen degradable protein (RDP) requirements, using soybean meal as protein supplement;
(B) 7.5 g/kg DM of urea inclusion, in partial substitution of soybean meal; (C) 15 g/kg DM

Table 1
Proportion of ingredients and composition of diets
Treatment (urea inclusion in diet)

0 7.5 15
Ingredients (g/kg DM)
Sugarcane 433.5 414.5 395.0
Ground corn grain 281.2 347.2 413.3
Soybean meal 257.0 201.7 146.1
Urea 0 7.5 15.0
Bicalcium phosphate 3.9 5.0 6.2
Limestone 9.4 9.4 9.4
Salt 5.0 5.0 5.0
Mineral mixture 10.0 10.0 10.0
Diet composition (g/kg DM)
Dry matter 618.2 638.5 640.3
Total nitrogen 25.87 26.13 25.94
Rumen degradable nitrogen (RDN)a 17.23 18.19 18.84
Rumen undegradable nitrogen (RUN)a 8.64 7.94 7.1
Acid detergent fiber 218.0 204.8 194.1
Neutral detergent fiber 317.6 303.4 295.6
Ether extract 52.2 50.3 51.3
Calcium 7.2 7.2 8.0
Phosphorus 4.6 4.4 4.5
Net energy for lactation (MJ kg−1 )a 6.40 6.40 6.40
Energy/rumen degradable nitrogen ratio 0.371 0.351 0.339
Ashb 49.1 47.0 43.0
a According to NRC (2001).
b Mineral mixture composition per kg: 180 g Ca, 90 g P, 20 g Mg, 20 g S, 100 g Na, 3000 mg Zn, 1000 mg Cu,
1250 mg Mn, 2000 mg Fe, 200 mg Co, 90 mg I, 36 mg Se, 900 mg F (maximum).
194 A.A. Aquino et al. / Animal Feed Science and Technology 140 (2008) 191–198

of urea inclusion, in partial substitution of soybean meal. Energy and protein levels of rations
were formulated according to NRC (2001), and were isoenergetic – 6.40 MJ net energy of
lactation/kg DM – and isonitrogenous – 160 g CP/kg DM.
Diets of each treatment were mixed as total ration and given to the cows twice a day,
right after milking. Animals were fed ad libitum allowing for 5% of orts.

2.3. Sampling and laboratory analysis

Daily milk samples were collected during the last 4 days of each experimental period.
Each sample was proportionally taken from each milking, 60% of the volume collected
in the first milking and 40% in the second. Milk samples were analyzed for urea by the
colorimetric-enzymatic method using Chemspec 150 (Bentley Instruments Inc., Chasca,
MN, USA), total nitrogen (TN) (AOAC, 1990; method 33.2.11; 991.20), non-casein nitrogen
(NCN) (Lynch et al., 1998), and non-protein nitrogen (NPN) concentrations (AOAC, 1995;
method 33.2.12; 991.21). In order to express the results as crude protein (CP), total nitrogen
values found in milk analyses were multiplied by 6.38 (Barbano and Clark, 1990). Milk
true protein (MTP) and casein concentrations were obtained by difference according to: CP
– EqNPN = true protein, and MTP – EqNNC = casein, respectively.
Diet samples were collected daily during the last 4 days of the experimental periods,
and a portion of each composite was dried at 100 ◦ C for 16 h to determine its DM content.
Remaining samples were air dried at room temperature for approximately 96 h, ground
through a 1 mm screen using a Wiley Mill (Arthur A. Thomas, Philadelphia, PA, USA) and
stored frozen at −20 ◦ C in a sealed plastic container for subsequent analyses. Immediately
after dry matter analyses (AOAC, 1990; method 934.01), samples were grinded and ana-
lyzed for crude protein (AOAC, 1990; method 988.05), ether extract (AOAC, 1990; method
920.39), ash (AOAC, 1990; method 942.05), calcium (AOAC, 1990; method 927.02) and
phosphorus. Concentrations of aNDF and ADFom exclusive of residual ash were measured
according to the procedures of Van Soest et al. (1991) and AOAC (1990; method 973.18),
respectively, with the use of a heat stable amylase but without sodium sulfite.

2.4. Statistical analysis

The averages of milk protein compositions were obtained during the last 4 days for
each cow in each period, excluding lost samples (8 out of 108). Data were analyzed using
the GLM procedure (Proc GLM; SAS® , Version 8.02; SAS Institute Inc., Cary, NC) to
account for treatment effects, animal within square, period and square. Effects of factors
were declared significant at P<0.05.

3. Results and discussion

The effect of dietary urea inclusion in total protein, equivalent of non-protein nitrogen
(EqNPN), non-casein nitrogen (NCN), true protein (TP), casein (CN), casein:true protein
ratio, and urea, as well as coefficient of variance, probability of linear (L) and quadratic
effect (Q), are shown in Table 2.
 A.A. Aquino et al. / Animal Feed Science and Technology 140 (2008) 191–198 195

Table 2
Effect of dietary urea inclusion on milk protein fractions
Variable Treatment Mean CV P
(% of urea inclusion in diet)
0 0.75 1.5 Linear Quadratic
Total protein (TP, g/kg) 33.9 32.0 32.7 32.8 11.78 0.228 0.075
Equivalent non-protein 2.6 2.6 2.6 2.6 9.99 0.542 0.589
nitrogen (g/kg)a
Non-casein nitrogen (g/kg) 6.8 7.2 7.4 7.4 13.89 0.217 0.823
True protein (g/kg) 31.3 29.5 30.2 30.4 12.81 0.255 0.092
True protein (g/kg total 923.2 919.3 919.6 920.7 1.40 0.542 0.589
protein)
Casein (g/kg) 26.5 24.8 25.3 25.5 12.88 0.117 0.084
Casein (g/kg total nitrogen) 795.5 772.7 772.3 779.9 3.13 0.100 0.321
Casein/true protein 0.86 0.84 0.84 0.85 3.22 0.220 0.500
Whey protein (g/kg) 4.3 4.7 4.9 4.6 22.13 0.349 0.889
Urea (mg/dl) 17.97 17.28 17.48 17.56 16.59 0.161 0.362
a NPN × 6.38.

The levels of urea inclusion in diets did not influence crude protein concentrations in milk
(P=0.228 for linear effect and P=0.075 for quadratic effect, Table 2). These results agree
with Christensen et al. (1993) that found no influence on milk yield and composition when
feeding dairy cows with proteins of different degradability and concentrations. Additionally,
the lack of effect on crude protein is also in agreement with Santos et al. (1998), who did
not report difference between milk composition from cows fed diets with different levels
of urea, soybean meal and fishmeal.
Susmel et al. (1995) found an increase on milk protein yield due to urea addition in
ration fed to dairy cows. Such results might be explained by the increase of microbial
protein synthesis, which led to an increase on milk and protein yield. In the conditions
of the present study, partial substitution of soybean meal by urea did not affect microbial
protein production capability, what could be explained by the fact that sugarcane and ground
corn contain enough easily fermentable organic matter allowing the microbes to capture
the extra N into microbial protein. Results of the present study are in disagreement with
Oliveira et al. (2001) and Silva et al. (2001) that detected negative linear effect on total milk
protein using 0, 7, 14 and 21 g/kg DM of urea inclusion in diets.
The energy/rumen degradable nitrogen ratios were 0.371, 0.351, and 0.339 for the inclu-
sion of 0, 7.5 and 15 g/kg DM urea. In the present study, substituting soybean meal for urea
generated RDP levels of 106.5, 111.4 and 116.2 g/kg in treatments 0, 7.5 and 15 g/kg DM
of urea inclusion, respectively. Considering these RDP levels, no differences were observed
among treatments on milk true protein concentration. Reynal and Broderick (2005) observed
a quadratic effect of RDP levels on milk true protein yield, and the maximum yield was
observed at 123 g/kg of RDP. The efficiency of nitrogen use decreased linearly, while N
excretion augmented with increasing levels of RDP. These results indicated that optimum
percentage of RDP for mid-lactation cows depends on the efficiency of nitrogen use.
It might be considered that the RDP levels used by Reynal and Broderick (2005) are
overestimated in comparison to the one recommended by NRC (2001), which is 9.2% of
RDP on diet DM. The results of the present study also differ from those reported by Roseler
196 A.A. Aquino et al. / Animal Feed Science and Technology 140 (2008) 191–198

et al. (1993) that observed that milk true protein could be increased by the usage of rumen
undegradable protein sources (RUP) due to higher amino acids availability in small intestine
for milk protein synthesis. In the present study neither urea inclusion nor the increase of
rumen degradable protein levels in diets affected the capacity to produce milk true protein.
Results for casein concentrations found in the present study are similar to the levels
reported by Bateman et al. (1999). Comparing the use of urea or soybean meal to different
sources of RUP and alfalfa as roughage, Bateman et al. (1999) did not found statistical differ-
ences among treatments for milk casein concentration. However, results of the present study
are in disagreement with Sampelayo et al. (1998, 1999), who showed that the degradable
fraction of dietary protein in lactating goats was the major factor responsible for variations
in casein concentrations.
Milk true protein:crude protein ratio, as well as casein:milk true protein ratio was not
influenced by urea concentrations in diets, which is similar to the results reported by Coulon
et al. (1998).
Milk urea nitrogen (MUN) concentrations have being largely used as indirect mea-
surement of nitrogen usage efficiency, since this parameter is highly correlated (r = 0.88) to
plasma urea nitrogen. High nitrogen concentrations may indicate an excessive protein nutri-
tion, which leads to unnecessary expenditures and environmental pollution, due to nitrogen
excretion (Jonker et al., 2002; Roseler et al., 1993). Milk urea concentrations measured in
this study are ranging accordingly to the normal minimum and maximum limits for lactating
Holstein cows (Meyer et al., 2004) – 12–18 mg/dl – and were not influenced by the effects
of urea addition in diets. Differently, Baker et al. (1995), studying degradability and RDP
and RUP relationships in diets fed to lactating cows, described a significant effect on milk
urea nitrogen and milk true protein. According to the conditions of the present study, diets
from all treatments supplied adequate amount of RDP, and partially substitution of soybean
meal by urea in ration did not influence milk concentration or its composition.

4. Conclusion

Based on the results of the present study, it can be concluded that for sugarcane based
diets, the addition of urea up to 15 g/kg DM in diets fed to mid-lactation cows did not alter
milk protein concentration, casein, whey protein or its non-protein fractions.

Acknowledgements

The authors thank the FAPESP, Fundação de Amparo a Pesquisa do Estado de São
Paulo, Brazil for the financial support (grants 03/01957-9), and Lucineia Mestieri and José
Franchini Garcia Moreno for technical assistance.

References

AOAC, 1995. Official Methods of Analysis, 16th ed. Association of Official Analytical Chemists, Arlington, VA.
AOAC, 1990. Official Methods of Analysis, 15th ed. Association of Official Analytical Chemists, Arlington, VA.
 A.A. Aquino et al. / Animal Feed Science and Technology 140 (2008) 191–198 197

Baker, L.D., Fergunson, J.D., Chalupa, W., 1995. Responses in urea and true protein feeding schemes for protein
of milk to different dairy cows. J. Dairy Sci. 78, 2424–2434.
Barbano, D.M., Clark, J.L., 1990. Kjeldahl method for determination of total nitrogen content of milk: collaborative
study. J. Assoc. Off. Anal. Chem. 73, 849–859.
Bateman, H.G., Spain, J.N., Kerley, M.S, Belyea, R.L., Marshall, R.T., 1999. Evaluation of ruminally protected
methionine and lysine or blood meal and fish meal as protein sources for lactating Holsteins. J. Dairy Sci. 82,
2115–2120.
Cameron, M.R., Klusmeyer, T.H., Lynch, G.L., Clark, J.H., Nelson, D.R., 1991. Effects of urea and starch on rumen
fermentation, nutrient passage to the duodenum, and performance of cows. J. Dairy Sci. 74, 1321–1336.
Christensen, R.A., Lynch, G.L., Clark, J.H., Yu, Y., 1993. Influence of amount and degradability of protein on
production of milk and milk components by lactating Holstein cows. J. Dairy Sci. 76, 3490–3496.
Coulon, J.B., Hurtaud, C., Remond, B., Verite, R., 1998. Factors contributing to variation in the proportion of
casein in cows’ milk true protein: a review of recent INRA experiments. J. Dairy Res. 65, 375–387.
Depeters, E.J., Cant, J.P., 1992. Nutritional factors influencing the nitrogen composition of bovine milk—a review.
J. Dairy Sci. 75, 2043–2070.
Emmons, D.B., Dubé, C., Modler, H.W., 2003. Transfer of protein from milk to cheese. J. Dairy Sci. 86, 49–
485.
Farrell, H.M., Jimenez Flores, R., Bleck, G.T., Brown, E.M., Butler, J.E., Creamer, L.K., Hicks, C.L., Hollar, C.M.,
Ng-Kwai-Hang, K.F., Swaisgood, H.E., 2004. Nomenclature of the proteins of cows’ milk—sixth revision. J.
Dairy Sci. 87, 1641–1674.
Hoover, C.W., Stokes, S.R., 1991. Balancing carbohydrates and proteins for optimum rumen microbial yield. J.
Dairy Sci. 74, 3630–3638.
Jonker, J.S., Kohn, R.A., High, J., 2002. Use of milk urea nitrogen to improve dairy cows diets. J. Dairy Sci. 85,
939–946.
Lynch, J.M., Barbano, D.M., Fleming, J.R., 1998. Indirect and direct determination of the casein content of milk
by Kjeldahl nitrogen analysis: collaborative study. J. AOAC 81, 763–774.
Magalhães, A.L.R., Campos, J.M.S., Valadares Filho, S.C., Torres, R.A., Mendes Neto, J., Assis, A.J., 2004.
Effects of replacing corn silage with sugarcane on production and ruminal metabolism of lactating dairy cows.
Braz. J. Anim. Sci. 33, 1292–1302.
Meisel, H., 2004. Multifunctional peptides encrypted in milk proteins. Biofactors 21 (1–4), 55–61.
Mendonça, S.D., Campos, J.M.D., Valadares Filho, S.D.V., Valadares, R.F.D., Soares, C.A., Lana, R.D., Queiroz,
A.C., Assis, A.J., Pereira, M.L.A., 2004. Intake, apparent digestibility, milk production and composition and
ruminal variables of dairy cows fed sugar cane based diets. Braz. J. Anim. Sci. 33, 481–492.
Meyer, P.M., Machado, P.F., Coldebella, A., Corassin, C.H., Cassoli, L.D., Coelho, K.O., Rodrigues, P.H.M.,
2004. Development of models to estimate milk urea nitrogen concentrations. J. Anim. Feed Sci. 13 (Suppl. 1),
527–530.
National Research Council, 2001. Nutrient Requirements of Dairy Cattle. Natl. Acad. Sci., Washington, DC.
Oliveira, A.S., Valadares, R.F.D., Valadares Filho, S.C., Cecon, P.R., Oliveira, G.A., Silva, R.M.N., Costa, M.A.L.,
2001. Intake, apparent digestibility, milk composition and production of lactating cows fed four non protein
nitrogen compounds levels. Braz. J. Anim. Sci. 30, 1358–1366.
Reynal, S.M., Broderick, G.A., 2005. Effect of dietary level of rumen-degraded protein on production and nitrogen
metabolism in lactating dairy cows. J. Dairy Sci. 88, 4045–4064.
Roseler, D.K., Ferguson, J.D., Sniffen, C.J., Herrema, J., 1993. Dietary protein degradability effects on plasma
and milk urea nitrogen and milk nonprotein nitrogen in Holstein cows. J. Dairy Sci. 76, 525–534.
Sampelayo, M.R.S., Amigo, L., Ares, J.L., Sanz, B., Boza, J., 1998. The use of diets with different protein sources
in lactating goats: composition of milk and its suitability for cheese production. Small Rumin. Res. 31, 37–43.
Sampelayo, M.R.S., Perez, M.L., Extremera, F.G., Boza, J.J., Boza, J., 1999. Use of different dietary protein
sources for lactating goats: milk production and composition as functions of protein degradability and amino
acid composition. J. Dairy Sci. 82, 555–565.
Santos, F.P., Huber, J.T., 1996. Quality of bypass protein fed to high-producing cow is important. Feedstuffs,
12–15.
Santos, F.A.P., Huber, J.T., Theurer, C.B., Swingle, R.S., Simas, J.M., Chen, K.H., Yu, P., 1998. Milk yield and
composition of lactating cows fed steam-flaked sorgum and graded concentrations of ruminally degradable
protein. J. Dairy Sci. 81, 215–220.
198 A.A. Aquino et al. / Animal Feed Science and Technology 140 (2008) 191–198

Silva, R.M.N., Valadares, R.F.D., Valadares Filho, S.C., Cecon, P.R., Campos, J.M.S., Oliveira, G.A., Oliveira,
A.S., 2001. Urea for dairy cows. 1. Intake, digestibility, milk production and composition. Braz. J. Anim. Sci.
30, 1639–1649.
Susmel, P., Spanghero, M., Stefanon, B., 1995. Nitrogen balance and partitioning of some nitrogen catabolites in
milk and urine of lactating cows. Livest. Prod. Sci. 44, 207–209.
Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for fiber, neutral detergent fiber, and nonstarch
polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597.