Effects of polymer coated slow-release urea on ruminal fermentation and nutrient total tract digestion of beef steers

The objective of this study was to evaluate the effects of polymer coated slow-release urea (SRU) in high-forage diets of beef steers on nutrient intake and digestibility, ruminal fermentation, microbial protein synthesis, and energy balance. Eight 24-mo-old rumen-fistulated castrated Nellore steers (average body weight = 418.0±40.0 kg) were used in a replicated 4 × 4 Latin square design. Animals were randomly distributed to receive one of the following diets: no urea inclusion; 1.0% inclusion of feed grade urea in the diet (dry matter [DM] basis); 1.0% inclusion of slow-release urea 1 in the diet (DM basis); and 1.0% inclusion of slow-release urea 2 in the diet (DM basis). Slow-release urea 2 had a similar composition to that of slow-release urea 1 and differed in that it contained 2.95% sulfur. A high-forage diet was provided (75% of total DM) and corn silage was used as the forage source. Diets with urea had increased crude protein (CP) intake, and CP and total digestible nutrients total tract digestion. Urea sources increased ruminal concentrations of ammonia nitrogen and acetate, and decreased butyrate concentrations. The polymer coated urea did not alter ruminal fermentation when compared with feed grade urea. Diets did not affect the energy balance of steers. Feed grade urea presented greater microbial protein synthesis than polymer coated slow-release urea. The partial replacement of soybean meal by 1% slow-release urea in a diet with 75% forage does not improve ruminal fermentation and microbial protein synthesis, and shows similar results as feeding feed grade urea


Introduction
Urea is the most common source of non-protein nitrogen (NPN) and is widely used in ruminant feeding because of its lower cost compared with true protein sources (e.g., soybean and cottonseed meal), representing an important source of rumen degradable protein (RDP).Urea supplementation is a common practice to meet the nitrogen requirement of animals fed high-forage diets.Ruminal fermentation of forage is slower than fermentation of non-fibrous carbohydrates (e.g., starch and sugars), and fermentation can be even slower when low-quality roughages are provided (Bergman, 1990).In the ruminal environment, dietary urea is rapidly hydrolyzed and metabolized into ammonia and CO 2 by urease, which increases ruminal ammonia concentrations during the first hour after feeding.However, when the rate of protein degradation exceeds the rate of carbohydrate utilization, large amounts of nitrogen can be lost as urea in the urine (Nocek and Russell, 1988).Furthermore, including feed grade urea in ruminant diets has potential negative effects due to the increased level of blood ammonia, and even death by ammonia toxicity if the diet is not appropriately mixed.
The goal of proper ruminant nutrition is to maximize microbial growth, improving the supply of amino acids to the small intestine and decreasing nutrient losses.Therefore, studies have been conducted aiming to obtain a greater synchrony between forage fermentation, urea hydrolysis, and ammonia utilization by ruminal microorganisms in order to improve the efficiency of NPN incorporation into microbial protein.Taylor-Edwards et al. (2009) demonstrated that polymer coated slow-release urea (SRU) can modulate (slower release and synchronized to form ammonia) the appearance of ammonia in the rumen environment when compared with feed grade diet.The SRU increased dry matter (DM) and nutrient intake of crossbreed steers fed a high-forage diet (Ribeiro et al., 2011) when compared with feed grade urea.Furthermore, Tedeschi et al. (2002) observed better feed conversion when growing steers were fed controlled release urea compared with urea in high-forage diets.
Considering the aforementioned facts, our hypothesis was that Nellore steers fed SRU would improve ruminal fermentation and microbial protein synthesis due to better utilization of ammonia.Therefore, this study was carried out to evaluate the effects of replacing soybean meal with polymer coated SRU in beef cattle diets on nutrient intake and total tract digestion, ruminal fermentation, microbial protein synthesis, and energy balance in Nellore steers.

Material and Methods
This study was approved by the Bioethics Committee of the School of Veterinary Medicine and Animal Sciences of Universidade de São Paulo, in accordance with the ethical principles of animal experimentation (protocol no. 1910/2010) Eight 24-mo-old rumen-fistulated castrated Nellore steers (average 418.0 kg±40.0kg) were randomly assigned to a replicated 4 × 4 Latin square design.The experimental periods consisted of 11 d of adaptation and 7 d of data collection.Steers were randomly assigned to the following diets: Control, no urea inclusion; Urea, 1.0% inclusion of feed grade urea (Reforce N ® , Petrobras Distribuidora S.A., Rio de Janeiro, RJ, Brazil) in the diet (DM basis); Slow-release urea 1 (SRU1: polymer coated urea synthetic polymer ® , Petrobras, Distribuidora S.A., Rio de Janeiro, RJ, Brazil), 1.0% inclusion of SRU1 in the diet (DM basis); and Slow-release urea 2 (SRU2: polymer coated urea synthetic polymer ® , Petrobras, Distribuidora S.A., Rio de Janeiro, RJ, Brazil), 1.0% inclusion of SRU2 in the diet (DM basis).Slow-release urea 2 had a similar composition to that of SRU1 and differed in that it contained 2.95% sulfur.Diets were formulated based on requirements described for an average daily gain (ADG) of 0.80 kg/d according to NRC (1996) (Table 1).The roughage:concentrate ratio of diets was set at 75:25, and corn silage was the roughage source.Diets were provided ad libitum once daily, at 7.00 h, as a total mixed ration.Steers were housed in a sand-bedded free-stall barn, with individual pens and forced ventilation during the entire experimental period.The feed and orts supplied to each steer were weighted daily and orts were restricted to 5-10% of intake on an as-fed basis, so as not to limit dry matte intake.
Feedstuffs and orts samples from each steer were collected daily from day 8 until day 11 and combined into composite samples.Fecal samples of each steer were collected twice daily from days 8 to 11 at 8.00 h and 16.00 h directly from the rectum, and composited into a single sample of each steer per period.All samples after collection were immediately frozen at −20 °C, for further analyses.
Feedstuffs, orts, and feces were dried at 55 ºC in a forced-air oven for 72 h and then ground to pass through a 2-mm screen (Wiley mill, Arthur H. Thomas, Philadelphia, PA, USA).Composite samples of feed supplied, orts, and fecal samples of each animal were analyzed for DM (method 95.15;AOAC, 2000); crude protein (CP), obtained by multiplying total nitrogen, determined using the micro Kjeldahl technique (method 984.13;AOAC, 2000), by a fixed conversion factor (6.25); and ether extract (EE), determined gravimetrically after extraction using petroleum ether in a Soxhlet apparatus (method 920.39;AOAC, 2000).The neutral (NDF) and acid (ADF) detergent fiber contents were determined using the methods described by Van Soest and Mason (1991).The NDF analyses were performed using α-amylase and without sodium sulfite in a fiber analyzer (model TE-149, Tecnal Equipamentos para Laboratorio Inc., Piracicaba, SP, Brazil).C -control; U -urea; SRU1 -polymer coated slow-release urea 1; SRU2 -polymer coated slow-release urea 2.
The estimate of total fecal excretion for each animal was determined based on concentration of indigestible ADF (iADF) as the internal marker, according to Casali et al. (2008).Dried and ground samples were placed in bags of non-woven fabric (100 g m 2 −1 ) with dimensions of 4 × 5 cm and incubated for 288 h in the rumen of two cannulated Nellore steers previously adapted to a similar diet to that of the present experiment.After removal from rumen, bags were washed in running tap water, dried at 55 ºC in a forced-air oven for 72 h, and then analyzed for ADF concentration as previously described.Digestibility was calculated using the level of iADF in feed (corrected for orts) and feces.
Rumen fluid samples (200 mL) were collected on day 11 of each period, at 0, 2, 4, 6, 8, 10, and 12 h after the morning feeding.Immediately after collection, rumen fluid pH values were determined using a pH meter (model MB-10, Marte Científica, Santa Rita do Sapucaí, MG, Brazil).Aliquots of samples were mixed with 20% metaphosphoric acid (0.25 Mol/L HPO 3 ) and then centrifuged at 7000 × g.The supernatant was stored at −20 °C in identified plastic tubes for subsequent analysis of volatile fatty acids (VFA).The remaining aliquots were mixed with sulfuric acid (0.5 mol/L H 2 SO 4 ) and stored at −20 °C for subsequent determination of ammonia nitrogen concentration (NH 3 -N) by the colorimetric phenolhypochlorite method (Broderick and Kang, 1980).
Volatile fatty acids were measured using a gas chromatograph (model GC-2014, Shimadzu, Tokyo, Japan) equipped with a capillary column (Stabilwax ® , Restek Corporation, Bellefonte, PA, USA).Gases used were helium (8.01 mL/min flow) as the carrier gas, hydrogen (pressure of 60 kPa) as the fuel gas, and synthetic air (pressure of 40 kPa) as the oxidizer gas.The steamer temperature was set at 220 °C and ionization detector flames at 250 °C.The separation column was set at 145 °C for 3 min, which was then raised by 10 °C/min up to 200 °C.
Energy values were calculated as follows: digestible energy (DE) intake = gross energy (GE) intake × GE digestibility (Havartine and Allen, 2006); net energy intake was calculated from DE using ME according to NRC (2001).Net energy for gain was calculated according to NRC (2001); and net energy available for maintenance was calculated as NE intake − NE gain.
Urine samples of 50 mL were collected from all animals on day 9 of each period, 4 h after the morning feeding by manual stimulation of the prepuce.The urine was filtered and 10 mL aliquots were immediately diluted into 40 mL of 0.036 N sulfuric acid to prevent bacterial lysis of purine derivatives and precipitation of uric acid.
Creatinine concentrations were determined with commercial kits (Laborlab ® , São Paulo, SP, Brazil), through a kinetic calorimetric enzymatic reaction using an automatic biochemistry analyzer (model SBA-200, Celm, São Caetano do Sul, SP, Brazil).Total daily urinary volume was estimated by dividing the daily creatinine urinary excretion by the creatinine concentration value in spot urine samples, as described by Chizzotti et al. (2007).The daily urinary excretion of creatinine was estimated from the proposition of 27.76 mg kg −1 BW for Nellore steers (Rennó, 2003).Thus, the total daily excretion of creatinine and creatinine concentration (mg/dL) in the spot urine sample and the total daily urine volume (L/d) were estimated.Urinary allantoin was determined using the modified colorimetric method of Fujihara et al. (1987), described by Chen and Gomes (1992).
Total excretion of purine derivatives (mmol/d) was calculated as the sum of allantoin and uric acid excreted in urine.The absorbed purine derivatives (PD abs , mmol/d) were calculated as follows: PD abs = (PD -0.385*BW 0.75 )/0.84, in which 0.385*BW 0.75 represents the endogenous excretion of PD (Chen and Gomes, 1992); and 0.84, the recovery of PD abs .The ruminal synthesis of nitrogen compounds (N mic, g N/d) was calculated based on absorbed purine derivatives, using the equation described by Chen and Gomes (1992): N mic = (70*PD abs )/(0.83*0.134*1,000),considering 70 as the N purine derivative content (mg N/mol); 0.134 as the purine derivatives N/microbial N ratio (Valadares et al., 1999); and 0.83 as the intestinal digestibility of microbial purines (Chen and Gomes, 1992).
Data were analyzed to check the normality of residuals and homogeneity of variance by using the UNIVARIATE procedure of SAS (Statistical Analysis System, version 9.1.3).Afterwards, data were analyzed by using the MIXED procedure of SAS, according to the model below: , in which y ijkl = observation on steer k given treatment i at period j in square l; α i = fixed effect of treatment i (i = 1 to 4); β j = fixed effect of period j (j = 1 to 4); = fixed effect of square l (l = 1 or 2); = random effect of steer within square (k = 1 to 8); and e ijk = random error associated with each observation.Ruminal fermentation data (pH, NH 3 , and VFA) were analyzed as repeated measures in the MIXED procedure of SAS (Statistical Analysis System, version 9.1.3)(0, 2, 4, 6, 8, 10, or 12 h post-feeding) and each variable was evaluated according to the model below: in which y ijkl = observation on steer k given treatment i at period j in square l; α i = fixed effect of treatment i (i = 1 to 4); β j = fixed effect of period j (j = 1 to 4); = fixed effect of square l (l = 1 or 2); = random effect of steer within square (k = 1 to 8); e ijk = random error associated with each observation of main plot (a); δ m = fixed effect of time m (m = 0, 2, 4, 6, 8, 10, or 12); αδ im = fixed effect of interaction between treatment i and time m; βδ jm = fixed effect of interaction between period j and time m; = fixed effect of interaction between Latin square l and time m; = random effect of interaction among steer k within each Latin square and time m; and e(b) ijklm = random error associated with each observation of subplot (b).To determine differences among diets, the following orthogonal contrasts were performed: (1) control vs. diets containing urea (U, SRU1 and SRU2); (2) urea vs. SRU1 and SRU2; and (3) SRU1 vs. SRU2.
Results of repeated measures analyses were subjected to three covariance structures: compound symmetric, first-order autoregressive, and unstructured.The covariance structure was chosen based on the smallest Akaike's information criterion values.Means were adjusted by LSMEANS and significance level was set at P≤0.05.

Results
Diets containing urea increased CP intake and CP and TDN total tract digestion when compared with control diet (Table 2; C1).Feed grade urea and polymer coated urea (SRU1 and SRU2) presented similar results for nutrient intake and total tract digestion.Moreover, SRU1 and SRU2 did not differ in intake and digestion of nutrients.
Urea sources increased ruminal concentration of ammonia and acetate; animals fed diets containing urea sources had a lower butyrate ruminal concentration (Table 3).No differences were observed among urea sources  (feed grade urea vs. polymer coated slow-release urea) in ruminal fermentation.However, SRU1 provided a higher propionate concentration when compared with SRU2.No interaction effect was observed.Experimental diets did not affect energy balance or energy efficiency utilization (Table 4).However, animals fed feed grade urea had greater microbial protein synthesis when compared with coated urea.

Discussion
Inclusion of urea in the animal diet, regardless of the source (SRU or U), resulted in higher CP intake and digestibility compared with control treatment (Table 2; C1).In this study, the higher CP intake for diets with urea was due to a higher concentration of CP in the diets of the animals when compared with control (15.21 vs. 14.36 g kg −1 of DM, respectively), since no difference (P<0.05) was observed for total DM intake.The higher CP digestibility is explained by the higher proportion of protein found after inclusion of urea.According to other authors (Taylor-Edwards et al., 2009;Highstreet et al., 2010), the protein fraction of the diet is more soluble and digestible.In a previous work, Lazzarini et al. (2009) stated that CP digestibility directly reflects the amounts of highly degradable nitrogen compounds in the diet.However, we did not observe statistically significant differences among the urea sources (SRU and U) for nutrient intake and digestibility (P>0.05).Previous studies (Puga et al., 2001;Galina et al., 2003;Galo et al., 2003;Xin et al, 2010) showed that SRU supplementation may improve the intakes of DM and nutrients when compared with U due to a higher activity of fibrolytic bacteria, resulting from an improved energy and N utilization by these microorganisms (Pinos-Rodríguez et al., 2010;Xin et al., 2010), with a consequent increase in the fiber fermentation (Taylor-Edwards et al., 2009;Xin et al., 2010;Holder et al., 2013).Lean et al. (2005) analyzed data from continuous culture fermenter studies and reported enhanced microbial CP synthesis and increased total tract digestion of CP and DM when a slow-release urea was used.In this work, we observed only a tendency (P = 0.072) of higher CP intake for animals fed the SRU diets.Lopez-Soto et al. (2014) demonstrated that the proportion of starch and fiber has a great influence on ruminal microbial growth and therefore on nutrient intake and digestibility for diets containing SRU and feed grade urea (FGU).The concentration of ruminal NH 3 -N increased for animals fed diets containing urea, but no interaction between time and diet was found.The NH 3 -N production rate is related to the solubility and NPN content of the degraded CP (NRC, 2001).Because the experimental treatments had a higher concentration of urea, which is totally degraded in rumen, and no differences were found in DMI, the higher level of ammonia in the rumen was expected.Similarly, Shain et al. (1998) and Milton et al. (1997) observed an increasing NH 3 -N ruminal concentration according to dietary urea inclusion.
Slow-release urea is hydrolyzed more slowly to ammonia than conventional urea, and could potentially be used more efficiently by rumen microorganisms and consequently decrease concentrations of ruminal NH 3 -N (Galo et al., 2003;Taylor-Edwards et al., 2009;Xin et al., 2010;Bourg et al., 2012).However, in the present study, NH 3 -N concentrations did not differ (P>0.05) between diets containing U and SRU.According to Owens and Zinn (1988), energy is a limiting factor in the microbial protein synthesis.López-Soto et al. (2014) demonstrated similar results when using different ratios of starch to ADF in diets with SRU and feed grade urea.In this study, the energy may have been a limiting factor to the growth of ruminal microorganisms for animals fed diets containing urea.Xin et al. (2010) noted a 15.6% greater microbial efficiency in SRU diet than diets with FGU and lower concentration of NH 3 -N.In a subsequent study with a roughage:concentrate ratio of 50:50, we observed that feeding SRU diets resulted in lower NH 3 -N concentrations when compared with U diets (data not published).
Animals fed diets containing urea had higher concentrations of acetate and lower butyrate concentration in the rumen.These results might be explained by a possible selective effect of urea sources on ruminal microorganisms.Some ruminal microorganisms, especially the fibrolytic bacteria, have a greater affinity for NPN.Therefore, supplementation with NPN sources may select these bacteria, and change the pattern of fermentation.Moreover, the NDF total tract digestion was approximately 38.5 g kg −1 higher for animals fed urea than for animals fed control diet, leading to high acetate concentrations.Xin et al. (2010) found similar results and suggested that higher acetate and lower butyrate concentrations in diets containing urea (FGU and SRU) resulted in lower conversion of acetate to butyrate in the rumen (Sharp et al., 1982;Sutton et al., 2003).Khattab et al. ( 2013) observed higher acetate concentration when feeding urea, and an increase in microbial protein synthesis.
Similar to other studies (Taylor-Edwards et al., 2009;Xin et al., 2010;Ding et al., 2014), when comparing SRU with FGU diets, we did not observe differences in concentration and molar proportions of VFA.According to Taylor-Edwards et al. (2009), replacing urea with SRU rarely affects any ruminal metabolite concentrations other than ammonia, at least in situations in which reduced ammonia concentrations presumably do not limit microbial growth.Our findings suggest that the urea source does not affect total production or ruminal concentrations of VFA.However, when comparing the SRU diets, feeding SRU1 resulted in a higher proportion of propionate and lower C2:C3, which may be due to the presence of sulfur (2.95%) in its composition.More detailed studies are necessary to elucidate this relationship.According to NRC (1996), sulfur supplementation is necessary when NPN is included in the diet due to the microbial synthesis of sulfur amino acids.
When analyzing the synthesis of microbial protein, we observed that animals fed SRU diets showed lower values of microbial CP and N mic (g d −1 ; Table 4; C2) than steers fed diets with FGU.These findings are opposed to what was expected.According to Russell et al. (2009), cellulolytic ruminal bacteria are unable to grow on other N sources in the absence of NH 3 and the stimulation of cellulolytic species by precursors of various N sources suggests a quantitative dependence on NH 3 -N-release rate for optimum growth (Cherdthongand Wanapat, 2010).Thus, the use of SRU should result in a better synchrony between the urea hydrolysis and ammonia utilization by ruminal bacteria (Holder el al., 2013), which would be demonstrated by higher N mic and microbial CP values for diets with SRU.Mehrez et al. (1977) stated that the ammonia concentration in the rumen needs to be 23.5 mg dL −1 for maximal fermentation rate.In this trial, the highest concentration of NH 3 -N (23.21 mg dL −1 ) was associated with the U group, which is close to values mentioned by Mehrez et al. (1977).However, in a later study of our group (data not published) conducted to evaluate the inclusion of 2% urea in diets with a roughage:concentrate ratio of 50:50, animals fed the U diet had higher concentrations of ruminal NH 3 -N (24.0 mg dL −1 ) compared with the SRU diets (SRU1: 20.7 mg dL −1 ; SRU2: 16.4 mg dL −1 ).On the other hand, SRU diets showed numerically higher values of microbial CP and N mic .Thus, microbial protein synthesis in the rumen may have been limited by the low availability of energy (López-Soto et al., 2014) and not because part of the NPN could leave the rumen without being converted to NH 3 by reducing its incorporation into microbial protein as other authors have suggested (Galo et al., 2003;Firkins et al., 2007).

Conclusions
The partial replacement of soybean meal by 1% slowrelease urea in a diet with 75% forage does not improve ruminal fermentation or microbial protein synthesis, and shows similar results as feeding feed grade urea to beef steer but without the potential hazards associated with feed grade urea.

Table 1 -
Ingredient and chemical composition of experimental diets

Table 2 -
Effects of different urea sources on nutrient intake and total tract digestion of Nellore steers 1 Estimated according to NRC (2001).

Table 4 -
Effects of different urea sources on efficiency of energy utilization, energy balance, and microbial protein synthesis of Nellore steers DE -digestible energy; NE L -net energy for lactation; EBWC -empty body weight change; NE G -net energy for gain; PD -purine derivatives; P abs , -absorbed microbial purines; N mic -microbial nitrogen; UV -urinary volume; ALA -allantoin. 1 NE L available for maintenance = NE L − NE G 2 NE G BW gain/digestible energy intake. R.Bras.Zootec.,45(2):63-70,2016