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Mazinani M, Naserian A. A, Mesgaran M. D, Valizadeh R. Effects of Adding Coated urea on In Vitro Gas Production of Dairy Cow. Biosci Biotech Res Asia 2018;15(2).
Manuscript received on : 28 April 2018
Manuscript accepted on : 10 May 2018
Published online on:  15-06-2018
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Effects of Adding Coated urea on In Vitro Gas Production of Dairy Cow

M. Mazinani1, A. A. Naserian2, M. Danesh Mesgaran2 and R. Valizadeh2

1Ruminant Nutrition, Ferdowsi University of Mashhad, Mashhad, Iran.

2Department of Animal Sciences, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.

Corresponding Author E-mail: mitra_mazinani@yahoo.com

DOI : http://dx.doi.org/10.13005/bbra/2638

ABSTRACT: In this experiment the effects of different urea products (urea, Paraffin-sulfur Coated Urea (PSCU) and controlled release urea product (Optigen, Alltech Inc., Lexington, KY)) on rumen fermentation were investigated in dependence of different diet sources by using in vitro techniques. The experiment followed a completely randomized design using four N-source treatmentes (urea, Paraffin-sulfur Coated Urea, Optigen and canola meal) in four diets (wheat straw+ %3 isonitrogenous of each N-source, barley grain+ %3 isonitrogenous of each N-source, barley grain+ molasses+ %3 isonitrogenous of each N-source and formulated TMR diets for dairy cow+ %3 isonitrogenous), the cumulative gas production (96 hours) influenced by diets and N-source treatments was different, which was higher gas production in formulated TMR diets for dairy cow and least gas production in wheat straw. The result indicated that Optigen (90.82) and then PSCU (90.81) the highest gas producer in the formulated TMR diets for dairy cow and the canola meal (69.04) and urea (69.43) had the least gas production in wheat straw (P<005). As a result, little difference between treatments for slow-release urea with control (canola) was observed in animal experiments. And therefore reducing feed costs and increasing the efficiency of the rumen microorganisms can be used NPN sources as a replacement for part of dietary protein.

KEYWORDS: Coated Urea; Urea; N-Source; Optigen

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Urea is a small organic compound that is very rich in N (44.96% N) that is used to supply degradable intake protein (DIP) to ruminants, which is cost-saving as compared with true protein feeds (Pfeffer et al. 2009; Taylor-Edwards et al. 2009; Calsamiglia et al. 2010; Cherdthong and Wanapat 2011). However, the amount of urea that can be used is limited because of the rapid hydrolysis to ammonia (NH3) in the rumen (Galo et al. 2003; Golombeski et al. 2006).Urea is broken down to ammonia in the rumen under the action of bacterial urease (Satter and Slyter, 1974). Microorganisms in the rumen are able to utilize the resulting ammonia to form amino acids which then become available to the host when microbial bodies are digested and absorbed post ruminally (Loosli et al., 1949).

The reasons for using urea over other sources of DIP is that urea N is cheaper on a per N basis than any other feedstuff and transportation and storage are cheaper and easier due to the concentrated nature of urea (McPherson and Witt, 1968). However, urea is used rather inefficiently by ruminants compared with other sources that contain true protein (Broderick et al., 2009), and this is due to the fact that the rate at which urea is degraded in the rumen is more rapid than the rate of utilization of the resulting ammonia by rumen bacteria leading to ruminal accumulation and absorption of ammonia and subsequent excretion of urea in the urine (Golombeski et al., 2006; Highstreet et al., 2010). Therefore utilizing urea as a DIP source may lead to excessive excretion of N in the urine. Nitrogen excretion from cattle operations has become a matter of increasing public concern over the last decade (VandeHaar and St-Pierre, 2006) and therefore reducing N excretion from cattle operations has become an increasing priority for ruminant nutritionists. One strategy for improving the utilization of urea by ruminants is to more closely time the fermentation of energy and the availability of ammonia in the rumen (Johnson, 1976). This may be done by increasing the degradability of the carbohydrates in the diet, or alternatively, by reducing the degradation rate of urea. A number of slow release urea products have been developed for this purpose (Deyoe et al., 1968; Fonnesbeck et al., 1975; Owens et al., 1980). Therefore, the objectives of this study was to develop Paraffin-sulfur Coated Urea and determine this Effects on in vitro gas production of dairy cows.

Materials and Method

Coating Urea in Rotary Drum

A rotary drum coater was used to coat the urea particles. The rotary drum coater was made from stainless steel and was 50 cm in diameter. Urea granules with a nitrogen content of 46%, was previously sieved to 1 mm in diameter were weighed and fed into the rotary drum rotating at 12 rpm. The materials selected as coating mixture were paraffin and sulfur15:1. Coating mixture was frequently applied onto the surface of the tumbling urea. Coated particles were collected after 20 min for analysis. (international patent number: A01K5/00, inventor: Mitra Mazinani)

Evaluate the Kinetics of NH3-N release

NH3-N release of each Optigen and PSCU were tested in sex liquid phase included (distilled water, TCA (Ph = 6.2), buffer solution, buffered rumen fluid, Free cell rumen fluid (centrifugation at 7000 rpm), Free cell buffered rumen fluid). Each of the tow N-sources was isonitrogenous (equivalent 20mg urea) and added to a 100-ml glass syringe. Ruminal fluid was obtained from three ruminally-fistulated Holstein cows fed a similar SBM basal diet. Then 30 ml of solution (consisting distilled water, TCA, buffer solution, buffered rumen fluid, Free cell rumen fluid or Free cell buffered rumen fluid) were pipette into each syringe followed by incubation in a water bath at 39°C. Three syringes for each treatment diet were incubated. At 0, 30 min and 2, 4 and 6 h time points, the syringes were taken out and Residual solid part was taken for determination of NH3-N release using the Kjeldahl N methods (AOAC, 2005).

In vitro gas production: To determine the gas production of each N-Sources (four N-Sources in triplicate) was tested in 4 different feed mixtures (straw + 3% N-Sources, barley flour + 3% N-Sources, barley flour + molasses as additives + 3% N-Sources, a dairy ration formulated to + 3% N-Sources), Each of the N-sources was isonitrogenous (equivalent 18 mg urea). Rumen fluid was obtained from two fistulated dairy cows fed twice daily with a diet containing forage (60%) and concentrate (40%). Equal volumes of ruminal fluid from each cow collected 2 h before the morning feeding squeized through four layers and mixed with McDougall (1948) buffer pre warmed to 39° C. The inoculums was dispensed (20 mL) per syringe into 100 mL serum syringe (containing of 200 mg sample per vial) which had been warmed to 39° C and flushed with oxygen free CO2. The syringes were sealed immediately after loading and were gently mixed and then mixed three times every 3h. Syringes for each time point, as well as blanks (containing no substrate), were prepared in triplicate. Triplicate syringes were removed after 2, 4, 6, 8, 10, 12, 16, 24, 36, 48, 72 and 96 h of incubation. The gas production was recorded at 2, 4, 6, 8, 12, 18, 24, 48, 72 and 96 h of incubation.

Cumulative gas production data were fitted to the model of Orskov and McDonald (1979). P=a+b(1-e−ct) that a=The gas production from the immediately soluble fraction (mL), b=The gas production from the insoluble fraction (mL), c=The gas production rate constant for the insoluble fraction (b), t=The incubation time (h) and P=The gas production at the time t.

Laboratory Analyses

CP analyzed with the Kjeldahl N methods (AOAC, 2005) at the Ferdowsi University of Mashhad and for ADF, NDF, and ash (AOAC, 2005).

Calculations and Statistical Analysis

Data were analyzed as a completely randomized design using a general linear model (GLM) procedure of SAS, with Duncan’s multiple range test used for the comparison of means.

Result and Discussion

According to figure 1, proportion of primary nitrogen to secondary nitrogen in the PSCU after 360 minutes of exposure to liquid was %48.26 ,compared to %72.19 for optigen. The residual nitrogen at all times except 10 minutes had the significant difference, and PSCU degradation Rate was higher. Result of NH3-N release of Optigen and PSCU in sex liquid phase showed in figure 2.

 Figure 1: The percentage of nitrogen remaining in optigen and Coated urea following liquid phase effect removal

Figure 1: The percentage of nitrogen remaining in optigen and Coated urea following liquid phase effect removal

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Based on Figure 2, the NH3-N release of Optigen and PSCU in all liquid phases except buffered rumen fluid Freicell was significantly different (P<005). The difference in the residual nitrogen under the effect of different liquid phases showed Optigen had greater NH3-N release in acidic conditions, while PSCU in the acidic condition and effect of  rumen microorganisms increased react and release. In both experiments have not brought up the results of urea culture as a source of urea was quick degradable (100% solution) and as soon as enter in any liquid phase, immediately dissolved (in less than 2 minutes).

 Figure 2: Amount of nitrogen remaining in optigen and Coated urea following removing time effect

Figure 2: Amount of nitrogen remaining in optigen and Coated urea following removing time effect

Click here to View figure

DiLorenzo and DiCostanzo (2007) evaluate the kinetics of  NH3-N release in bath culture. they showed at 6 h, deamination from SBM protein begins to be significant, as NH3-N accumulates relative to the Blank (p=0.02). from 8 to 24 h, Urea led to greatest (P<005) NH3-N release, and greater (P<001) NH3-N release when compared to that elicited by NTS (Balchem Encapsulates, New Hampton, NY) at all times. From 4 to 24 h, biuret had the lowest (P<005) NH3-N concentrations of all NPN source, this could be do to a reduced biruetase activity in the rumen since the rumen fluid source steer was not adapted to biuret. Nitrogen utilization by rumen microorganisms can be reflected by ruminal ammonia-N concentration (Hungate, 1966). Prokop  and Klopfenstein (1977), found that slow-release urea (combination of urea and formaldehyde) could decrease ruminal ammonia-N concentration by 25.3% compared to urea. A similar result was found in the report of Galo (2003), in which urea release from a polymer-coated urea was 83% as extensive as uncoated urea after 1 h incubation with distilled water. Cass and Richardson (1994) made a comparison in an in vitro study and observed that a urea-calcium combination produced slower ammonia nitrogen release rate than regular urea. Ammonia-N concentrations began to increase at 8 h for the FGU diet, which indicates that bacterial autolysis may occur. Based on this results, urea is often degraded rapidly in the rumen by the action of urease and the resulting ammonia supply may exceed the capacity of rumen bacteria to assimilate it into amino acids (Huber and Kung, 1981). This rapid release of ammonia may result in inefficient N utilization in the rumen. Therefore slow-release urea improve ammonia assimilation in the rumen.

Table 1: Diet composition and chemical analysis of different dietary treatments (% DM)

Item Control Urea Optigen Coated urea
Diet composition (% DM)
Alfalfa 40 40 40 40
Wheat straw 5 5 5 5
Corn 27.5 27.5 27.5 27.5
Canola meal 17.5 11 10.95 10.8
Wheat grain 8 14 14 14
Calcium carbonate 0.6 0.6 0.6 0.6
Salt 0.5 0.5 0.5 0.5
Urea 0 0.5 0 0
Coated urea 0 0 0 0.7
Optigen 0 0 0.55 0
Min-Vit Mix2 0.9 0.9 0.9 0.9
Chemical analysis (% DM)
Dry matter 90.4 90.32 90.31
Organic matter 80.2 80.46 80.43
Crude protein 15.54 15.2 15.05
Neutral detergent fibre 37 39.9 39.88
Acid detergent fibre 21.4 21 21
 Ash 10.2 9.86 9.88

1Treatments consist: 1) control (canola meal)., 2) urea (0.5% urea)., 3) optigen (0.55% optigen)., 4) Coated urea (0.7% Coated urea)

2Each kg contained: Vit A, 500000IU; Vit D3, 100000 IU; Vit E, 100mg; Ca, 190000mg; P, 90000mg; Na, 50000mg; Mg, 19000mg; Fe, 3000mg; Cu, 300mg; Mn, 2000mg; Zn, 3000mg; Co, 100mg; I 100mg; Se, 1mg; Antioxidant (B.H.T) 3000mg.

Table 2: Gas production in dietary samples containing  different nitrogen sources1

Item Sample Controll Urea Optigen Coated urea SEM P-value
Gas production (96 h) [ml/0.5 g DM substrate] Straw 69.04b 69.43b 72.63b 76.62a 1.41 <0.0056
Gas production (96 h) [ml/0.5 g DM substrate] Barely flour 72.04 72.84 74.27 75.41 2.67 NS2
Gas production (96 h) [ml/0.5 g DM substrate] Barley flour + molasses 74.17b 80.48a 75.97b 76.93b 0.86 <0.02
Gas production (96 h) [ml/0.5 g DM substrate] Ration formulated to dairy cow 91.81 91.04 90.82 91.19 1.39 NS
Smalle chaine fatty acids (mmol) Straw 0.271b 0.27b 0.28b 0.30a 0.006 <0.0056
Smalle chaine fatty acids (mmol) Barely flour 0.28 0.28 0.29 0.29 0.01 NS
Smalle chaine fatty acids (mmol) Barley flour + molasses 0.29b 0.31a 0.29b 0.30b 0.001 <0.02
Smalle chaine fatty acids (mmol) Ration formulated to dairy cow 0.36 0.36 0.36 0.36 0.006 NS
Metabolizable energy (MJ/kg) Straw 12.00c 12.13cb 12.63b 13.26a 0.22 <0.0045
Metabolizable energy (MJ/kg) Barely flour 12.49 12.69 12.91 13.09 0.42 NS
Metabolizable energy (MJ/kg) Barley flour + molasses 12.83b 13.91a 13.10b 13.46b 0.05 <0.02
Metabolizable energy (MJ/kg) Ration formulated to dairy cow 15.68 15.64 15.52 15.58 0.22 NS
Organic matter digestibility (%) Straw 77.85c 78.75cb 81.93b 85.88a 1.40 <0.0043
Organic matter digestibility (%) Barely flour 81.18 82.50 83.92 85.04 2.65 NS
Organic matter digestibility (%) Barley flour + molasses 83.28b 90.11a 84.98b 87.26b 0.34 <0.02
Organic matter digestibility (%) Ration formulated to dairy cow 101.09 100.79 100.08 100.45 1.38 NS
Organic matter digestibility in DM (%) Straw 71.93c 73.00cb 75.94b 79.61a 1.29 <0.0037
Organic matter digestibility in DM (%) Barely flour 78.83 80.19 81.57 82.66 2.57 NS
Organic matter digestibility in DM (%) Barley flour + molasses 80.20b 84.44a 79.63b 81.76b 0.32 <0.02
Organic matter digestibility in DM (%) Ration formulated to dairy cow 90.78 90.86 90.22 90.55 1.24 NS

1Means within same row with different superscripts differ(P<0.05).

2NS: not significant

Gas Production

Results showed the cumulative gas production (96 hours) influenced by diets and N-source treatments was different, which was higher gas production in formulated TMR diets for dairy cow and least gas production in wheat straw. The result indicated that Optigen (90.82) and then PSCU (90.81) the highest gas producer in the formulated TMR diets for dairy cow and the canola meal (69.04) and urea (69.43) had the least gas production in wheat straw (P<005) (table 2). Since the in vitro gas production technique has been used as a measurement of ruminal degradation of feed (Menke and Steingass 1988; Poungchompu et al. 2009) or hay (Karabulut et al. 2007), high gas production indicated high digestibility of substrates. In the current study, it was found that slow-release urea treatments had higher gas production. That was agree to other researches.


In this trial Optigen and PSCU were used as slow release urea. Rate of NH3-N release in Optigen as shown in figure 1,was %72.19 after 6 hours in bath culture It can be concluded, according to time that each ingredient remain in gastrointestinal tract, a large part of Optigen without being used, be excreted. PSCU had faster release rate and after 6 hours, NH3-N remaining was 48.26 percent. As a result, degradation rate should be considered as indicators to identify the sources of slow release urea.

In conclusion, final goal in dairy farms is maintained milk production and animal health. These experiments demonstrate that SRU can be utilized as an N supplement to modulate the appearance of N in the rumen and can provide equal performance to urea supplements without the potential hazards associated with feed-grade urea, and decrease feed cost and milk production price.


We gratefully acknowledge the authorities of the branch of Animal Sciences at the Ferdowsi University of Mashhad for their cooperation. This work was conducted as a Ph.D. thesis in Animal Sciences at Ferdowsi University of Mashhad (Iran). The authors gratefully thanks to Ferdowsi University of Mashhad for their endorsement, and they also especially like to appreciate Prof. A. Naserian for their comments.


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