Levels of nitrates in a urea fertilized Kikuyu (Cenchrus clandestinus (Hochst. ex Chiov.) Morrone) pasture on the high plains of Antioquia, Colombia/Niveles de nitrato en pasto Kikuyo (Cenchrus clandestinus (Hochst. ex Chiov.) Morrone) fertilizado con urea en el antiplano de Antioquia, Colombia/Niveles de nitrato en pasto Kikuyo (Cenchrus clandestinus (Hochst. ex Chiov.) ...
In Colombia, urea is extensively applied as a fertilizer on pastures used by dairy cattle (15). However, when crude protein content exceeds 22%, the excess nitrogen tends to buildup as nitrate salts that upon ingestion by the ruminant can be converted to potentially toxic levels of nitrites in the rumen (2,4,5,12). Most cattle extension services of North American Agricultural Colleges have some type of recommendations for managing forages with high nitrate content. In spite of the lack of agreement with regards to the levels of nitrate that may cause adverse health effects in cattle, almost all services agree that pastures with concentrations above 1.5% (on a dry matter basis for N[O.sub.3] ion) are potentially lethal, and their consumption should be avoided. Between 0.8-1.5% N[O.sub.3], the common recommendation is to limit consumption by mixing with other sources of feed. Although there is consensus among the toxicologists of the American Board of Veterinary Toxicology (Villar 2014, personal communications) that levels of N[O.sub.3] below 1% are safe, a 0.5% margin of safety is usually added due to the wide variation that can be found in the same bale of hay, resulting in a recommended cutoff level of 0.5% for all cattle rations.
Kikuyu grass (Cenchrus clandestinus) constitutes around 90% of pastures on the high tropics of Antioquia (3). Despite being a good fodder plant for the tropics, it has been associated with acute poisonings of cattle in Australia and South Africa (1). So far, the toxic principle of such poisonings remains unidentified, but a toxin known as "wortmannin" produced by the fungus Fusarium tolurosum is the main suspect (14). Studies in South Africa showed that, when the proportion of stem tissue is high in fertilized old Kikuyu pastures, the concentration of nitrates could reach potentially dangerous levels for cattle above 1% DM (9). Considering the high levels of fertilization that kikuyu pastures receive in many areas of Antioquia, experimental studies that mimic some common fertilization practices are warranted to evaluate the potential accumulation of nitrate to hazardous levels. Thus, the objective of this study was to determine the effects of four levels of nitrogen fertilization on dry matter yield and the potential for nitrate accumulation in kikuyu grass.
Materials and methods
The experiment was carried out at the Agricultural Station of the National University of Colombia at Santa Elena (Antioquia), located at 2500 meters of altitude, and considered a highly humid tropical climate. The field was a long-established kikuyu pasture and Holstein cattle had been grazing with vacant periods of 42 days to allow recovery of biomass. Sixteen plots of 12 [m.sup.2] were measured in the paddock and assigned to receive topdressing urea with one of four treatments per plot as follows:
T0: no fertilization (control),
T1: recommended fertilization (50 kg N/ha)
T2: 2-fold recommendation (100 kg N/ha)
T3: 4-fold recommendation (200 kg/ha)
Each treatment was replicated four times at the end of the growing period between December 2012 and June 2013. The collection of grass samples was performed at the end of the 42 day growing period and meant to represent cattle grazing exposure. Whole grass (leaves and stems) were hand mowed from each plot and used to determine dry biomass and chemical analysis. Dried kikuyu samples were sent to the Veterinary Diagnostic Laboratory of Iowa State University for chemical analysis. Nitrate and nitrites were simultaneously determined by ion chromatography as described by Kissner and Koppenol (2005). Briefly, 2 g of ground samples were extracted in duplicate into water at a 1:10 dilution factor. The centrifuged extracts were again diluted using another 1:10 dilution factor or more (1:200) when concentrations were too high. The analysis was performed using a Dionex ICS 3000 with a AS22 4mm guard and analytical column combination. The eluent was 4.5 mM sodium carbonate/1.4 mM sodium bicarbonate. A standard curve with potassium nitrate containing 0.1, 1, 5, 10, 20, 50, and 100 ppm was used for comparison.
Mean values for each urea treatment were compared by analysis of variance for repeated measures (Statistica, StatSoftware, version 7). All data were checked for normal distribution by the Shapiro--Wilks' W-test. Homocedasticity was verified with the Levene's test, and the data were log transformed when these conditions were not met.
There was a statistically significant (p [less than or equal to] 0,05) greater dry matter yield in all urea treated plots compared to untreated ones, and the combined yields of all experimental periods for each application rate is shown in figure 1. Unexpectedly, no correlation was observed between biomass production and increased application of urea at any of the four experimental periods.
With respect to the effect on nitrate concentrations, there was a significant (p [less than or equal to] 0,05) effect of urea application rates and a positive dose vs time interaction (p [less than or equal to] 0,05) on the overall concentration of nitrate in Kikuyu grass samples. When all four month samples for each urea application rates were combined, the mean [+ or -] SEM concentration of nitrates significantly (P [less than or equal to] 0,01) increased from 369 [+ or -] 216 ppm with no fertilization, to 881 [+ or -] 208 ppm at the highest rate of 200 kg N/ha (Figure 2). However, there was a large variation of concentrations within each treatment, and only 1 sample in a plot receiving urea at 100 kg N/ha barely exceeded the 5000 ppm threshold limit for making recommendations of limited consumption.
The results of this study disproved a common belief that over-fertilization with urea on a typical old kikuyu pasture in Antioquia could elevate nitrate concentrations to potentially toxic levels. All nitrate levels obtained in this experiment were very low. A similar study conducted in South Africa in old kikuyu pastures, but using limestone ammonium nitrate as the source of nitrogen, showed that with applications of 100 and 200 kg N/ha, nitrate concentration in complete tiller cuts were below 1000 ppm, and just at the highest level of 500 kg N/ha, the concentrations exceeded 3000 ppm (9). However, their study showed that when tillers were separated into leaves and stem tissue, mean concentrations of 1.6% (=16.000 ppm) were obtained in the stems of fertilized samples. Although such high concentrations in stem tissues could initially raise alarms, stem tissue only constitutes a relatively low fraction of the dry mass of the tiller. Assuming the worst case scenario that animals were exposed to such high concentrations of nitrate in a kikuyu pasture, the speed of consumption under grazing conditions would be too slow to cause a rapid increase of nitrites in the rumen. Experiments in the 70's showed that at equal nitrate intakes, the higher speed of consumption with hay, as compared with fresh roughage, was enough to form toxic concentrations of nitrite in the rumen that would cause methemoglobin formation in the blood (6, 7). In addition, those studies showed that the speed at which nitrates are released in the rumen is much faster with hay than fresh grass, which again, makes grazing conditions more unlikely to reach toxic nitrate concentrations in the rumen at any given time.
With regards to the effect on DM production, our results showed that the application of urea caused a significant increase from 1194 kg DM/ha with no fertilization, to overall means of between 1867 and 2249 kg DM/ha with 50 to 200 kg N/ha (Figure 1). However, no greater production was attained with levels above 50 kg N/ha, suggesting that plant demands for nitrogen were already adequate and further application rates would result in nitrogen environmental losses. Furthermore, in a previous study conducted on the same field plots of the present study (15), it was concluded that the effect of cutting age and nitrogen fertilization had no significant effects on the nutritional quality of the kikuyu grass, reinforcing the need to base any fertilization program on specific demands for each particular field and type of forage. The overall kikuyu mass production attained here was similar ([greater than or equal to] 2000 kg DM/ha) to that reported for the high Colombian tropics, following recovery methods such as mechanical soil loosening and treatment with compost or other sources of fertilization (10,13).
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(Recibido: 1 de abril, 2014; aceptado: 6 de junio, 2014)
Jhon Didier Ruiz Buitrago  *, MV, MSc, PhD; David Villar Argaiz , MV, PhD; Hector Jairo Correa , Zoot. PhD; Jorge Mario Norena Grisales , Ing. Agr, Econ, Esp; Manuela Roldan , Est. MVZ; Juan Camilo Rios , Est. MVZ.
* Autorpara correspondencia: Jhon Didier RuizBuitrago. Calle 10aNo 22-04. Medellin Colombia. firstname.lastname@example.org
 Grupo de Investigaciones en Ciencias de los Animales (INCA-CES), Facultad de Medicina Veterinaria y Zootecnia, Universidad CES, Medellin, Colombia.  Grupo de Investigacion Vericel. Facultad de Ciencias Agrarias. Universidad de Antioquia. Medellin, Colombia.  Grupo de Investigacion Interacciones Nutricionales, Metabolicas y Reproductivas en Rumiantes. Facultad de Ciencias Agrarias de la Universidad Nacional, sede Medellin. Colombia.
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|Title Annotation:||articulo en ingles|
|Author:||Buitrago, Jhon Didier Ruiz; Argaiz, David Villar; Correa, Hector Jairo; Grisales, Jorge Mario Norena|
|Publication:||Revista CES Medicina Veterinaria y Zootecnia|
|Date:||Jan 1, 2014|
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