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- rotein 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 (Magalh˜aes et al., 2004). Mendonc¸a 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 concerning 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.
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 periods (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/kgDM of urea inclusion, in partial substitution of soybean meal; (C) 15 g/kgDM 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/kgDM – 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 1mm screen using aWiley 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 analyzed 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.
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 milkprotein using 0, 7, 14 and 21 g/kgDM of urea inclusion in diets.
The energy/rumen degradable nitrogen ratios were 0.371, 0.351, and 0.339 for the inclusion of 0, 7.5 and 15 g/kgDM 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/kgDM 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 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 ofRUP and alfalfa as roughage, Bateman et al. (1999) did not found statistical differences 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 measurement 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 nutrition, 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.
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/kgDM in diets fed to mid-lactation cows did not alter milk protein concentration, casein, whey protein or its non-protein fractions.
The authors thank the FAPESP, Fundac¸ ˜ao de Amparo a Pesquisa do Estado de S˜ao Paulo, Brazil for the financial support (grants 03/01957-9), and Lucineia Mestieri and Jos´eFranchini Garcia Moreno for technical assistance.
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This article was originally published by Elsevier on Animal Feed Science and Technology Journal, Vol 140, 191-198, 2008. Engormix.com thanks the aurthor for this contribution.