Introduction
The use of sugarcane (Saccharum spp L.) in the in natura form to feed cattle is an established practice among Brazilian cattle ranchers. However, the daily cutting of sugarcane is burdensome and causes logistical and operational problems owing to the daily demands of labor. Thus, sugarcane is generally used as silage to feed cattle (Rigueira et al., 2018). However, when sugarcane is ensiled, it presents a fermentative pattern characterized by high ethanol production and loss of soluble carbohydrates, which is mediated by yeasts; reducing its nutritional value and losing dry matter (DM) (Moreira et al., 2014; Roth et al., 2016).
Urea is often used as an additive to silage. This is based on the transformation of urea into ammonia, which reacts with water to form ammonium hydroxide, raising the pH, and inhibiting the metabolism of undesirable microorganisms such as yeasts (Kung Junior et al., 2003). The buffering effect of ammonium hydroxide on silage pH can be advantageous, although this depends on the extent to which it is able to delay pH decrease. If the buffering effect is moderate, then there is greater competition between lactic acid bacteria and yeasts, since the latter are predominant in fermentation at pH values below 4. However, if the buffering effect is large, then development of undesirable microorganisms that act at higher pH is favored, causing forage deterioration.
Recently, a product termed ‘protected urea’ has been developed. It generates a controlled release of ammonia and reduces the buffering effect caused by conventional urea in addition to allowing higher concentration to be used at the time of ensilage.
Therefore, the objective of this work was to compare the fermentative characteristics of adding protected or conventional urea to sugarcane silage and to assess the nutritive value of the silage.
Materials and methods
Ethical considerations
The experiment was approved by the Ethics Committee for Animal Experimentation of Uni versidade Estadual de Montes Claros (number 155/2018). The study was performed at the De partamento de ciências agrárias da Universidade Estadual de Montes Claros (UNIMONTES), Campus Janaúba-MG, Brazil (GPS coordinates: latitude 15°52′38″ S, longitude 43°20′05″ W).
Experimental design
A completely randomized 2×2+1 factorial design was used, based on two types of urea (conventional and protected), two urea concen trations (0.5 and 1% of the natural matter), and a control (sugarcane silage with no additives). Six replicates were used.
Procedures
The sugarcane variety used was IAC 86- 2480, obtained from the experimental farm of UNIMONTES. The forage was cut manually. One year after the last cut, the stalks were chopped using a stationary forage harvester (Nogueira, São João da Boa Vista, SP, Brazil) with the knives adjusted to obtain 1 to 2 cm particle size. Four 20 kg piles of fresh sugarcane were harvested, protected or conventional urea was added at the desired proportions, and then the mixture was homogenized.
The silages were prepared in PVC silos, 50 cm in height and 10 cm in diameter, with a Bunsen valve. At the bottom of the silos, 0.4 kg of dry sand was added to drain the effluents, and a layer of foam to prevent contact between the silage and the sand. The experimental set, consisting of silo plus a lid, sand, and foam, was weighed to obtain the empty weight of the silo. Subsequently, the mixtures to be ensiled were placed into the silos and compacted to a density of 550 kg m-3 (Ruppel et al., 1995). The silos were sealed, stored at room temperature (25 °C), and opened only after 60 days. Before opening, the silos were reweighed to quantify gas losses.
After removal of the silage, the experimental set containing silo plus lid, sand, and foam were reweighed to quantify the produced effluent (Jobim et al., 2007).
After the silage was removed from the silos, it was homogenized; one part was pressed with a hydraulic press (Bovenau®, Rio do Sul, SC, Brazil) to extract silage juice. The pH of this liquid was determined using a potentiometer (Tecnal®, Piracicaba, SP, Brazil), and the level of ammoniacal nitrogen (N-NH3) was estimated by distillation with magnesium oxide and calcium chloride using a boric acid receptor solution and titration with 0.1 N hydrochloric acid (AOAC, 1980). The remainder of the silage was dried in a forced ventilation oven at 55 °C for 72 h and ground in a mill (Thomas Model 4 Wiley; Thomas Scientific, Swedesboro, NJ, USA) with 1 mm sieve screens for chemical analysis, and 5 mm for in situ incubation.
Analyses were carried out according to the analytical procedures of the National Institute of Science and Technology in Animal Science (INCT-CA; Detmann et al., 2012). Dry matter (DM), crude protein (CP), ether extract (EE), neutral detergent fiber (NDF), acid detergent fiber (ADF), and lignin were assessed.
To evaluate ruminal degradation kinetics, four 450 ± 50 kg crossbred steers were used. All the animals had a gastric cannula. The animals received 3.0 kg concentrate in two equal portions in the morning and afternoon, as well as diet based on cane silage. The in situ degradability technique described by Casali et al. (2009) was performed using 7.5 × 15 cm non-woven fabric bags with an approximate porosity of 60 μm. The number of samples was determined from the ratio of 20 mg DM cm-2 of bag surface area (Nocek, 1988).
The bags were placed in 20 × 30 cm fillet bags along with 100 g lead weights. The fillet bags were tied with a nylon thread, leaving a length of 1 m so that the bags could move freely in the solid and liquid phases of the rumen. The fillet bags were then deposited in the ventral sac region of the rumen with the end of the nylon thread remaining attached to the cannula for 0, 6, 12, 24, 48, 72, 96, 120 and 144 h in reverse order, i.e., starting with the duration of 144 h. The 0 h time samples were inserted into the rumen for 5 min. Subsequently, all samples were collected and washed in ice water to stop ruminal fermentation. Afterward, the samples were placed in greenhouses at 55 °C for 72 hours and then cooled in a desiccator and weighed.
The residues remaining in the non-woven fabric bags following collection from the rumen were analyzed for DM and NDF contents. The percent degradation was calculated by the proportion of food remaining in the bags after ruminal incubation, and NDF was analyzed according to the methods proposed by Van Soest et al. (1991) without the use of α-amylase. The obtained data were adjusted to a non-linear regression by the Gauss-Newton method (Neter et al., 1985) using the SAS software (SAS, 2008), according to the equation proposed by Ørskov & Mcdonald (1979): Y= a+b (1-e-ct), where Y = is the cumulative degradation of the nutritional component analyzed after time t; a = the degradation curve intercept when t = 0, which corresponds to the water-soluble fraction of the analyzed nutrient component; b = the degradation potential of the water-insoluble fraction of the analyzed nutrient component; a+b = the degradation of the analyzed nutritional component when time is not a limiting factor; c = the rate of degradation per fermentative action of b; and t = incubation time.
Once calculated, the a, b, and c coefficients were applied to the equation proposed by Ørskov & McDonald (1979): ED= a+ (bxc/c+k), where ED = is the effective degradability of the nutritional component, and k = the feed passage rate. The particle passage rate through the rumen was estimated to be 5% h-1, as suggested by AFRC (1993).
Statistical analysis
A completely randomized design in a 2 x 2 +1factorial arrangement with two urea types (conventional and protected) in two concentrations (0.5 and 1% of the natural matter) plus one control treatment (sugarcane silage with no additives) and six replicates was used for fermentation traits and chemical composition variables. The data were submitted to analysis of variance using PROC GLM of SAS, version 9.2 (SAS Institute, Inc., Cary, NC, USA). When the "F" test was significant for the treatments, urea concentrations and types were analyzed by t test. For all tests, α=0.05 was used. The mathematical model used was Yijk = μ + τ i + doses j + τ i x doses j + ɛijk, where: Yijk is the observation ijk; μ, the overall mean; τ i, the effect of the treatment urea types, with i = 1 and 2; concentrations, the effect of urea concentrations; the random effect k; τ i x concentrations j, the interaction effect of urea types i and concentrations j; ɛijk is the random error with mean 0 and variance σ2.
For the in situ degradability variables, a split-plot randomized complete block was used, with five treatments (plots), nine incubation times (subplots), and four blocks. The different animal weights were the blocking factor. Data were tested via analysis of variance and regression using the MIXED procedure of SAS, at α=0.05, according to the model: Yijk = μ + τ i + Time j + τi x Time j + ɛijk, where: Yijk is the observation ijk; μ, the overall mean; τ i, the fixed effect of the treatment applied to the plot, with i = 1, 2, 3, 4 and 5; Time, fixed effect of incubation time j to subtract; the random effect of animal k; Ti x Time j, the interaction effect of treatment i and time j; ɛijk, the random error with mean 0 and variance σ2. The treatment means were compared to the control by Dunnett’s test (α=0.05).
Results
The DM contents of the silages did not differ with the addition of urea. The pH values were within a suitable range for forage conservation, with a difference only for the silage with 1% conventional urea compared with that of the control silage (p<0.05; Table 1).
1Type, effect of urea type; Concentration, effect of urea concentration; TxC: interaction effect between type and concentration of urea; AxCTL: effect of the additive compared to the control.
* and ** significant at the level of 5 or 1% significance, respectively.
* Means followed by an asterisk differ from the control by Dunett’s test (p<0.05).
Means followed by different superscript upper-case letters (A, B) within the same column, and different superscript lower-case letters (a, b) within the same row, are significantly different by the t test (p<0.05).
SEM - standard error of the mean.
The treatments with 1% urea had a higher fraction of N-NH3 than both the control and the 0.5% treatment (p<0.05). In relation to urea type, conventional urea showed higher N-NH3 fraction than that of protected urea (p<0.05). Effluent losses were not influenced by the addition of urea (p>0.05). Lower gas losses were observed in all urea treatments when compared with the control silage (p<0.05; Table 1).
There was a significant interaction between dose and type of urea for crude protein (p<0.05), and all treatments differed from the control (p<0.05). The EE content was higher for silages with 1% urea (p<0.05). Regarding the different types of urea, protected urea showed the highest levels (p<0.05; Table 2). Reductions in NDF, ADF, and lignin were observed in the fibrous fraction compared with that in the control treatment (p<0.05; Table 2). All treatments differed from the control for NDF (p<0.05). For ADF, only silage with 1% conventional urea differed from the control; for lignin, all treatments with 1% urea, regardless of type, differed from the control. There was no interaction effect between type and dose of urea (p>0.05).
1Type, effect of urea type; Dose, effect of urea dosage; TxC: interaction effect between type and dose of urea; AxCTL: effect of the additive compared to the control.
* and ** significant at the level of 5 or 1% significance respectively.
* Means followed by an asterisk differ from the control by Dunett’s test (p<0.05). Means followed by different superscript upper-case letters (A, B) within the same column, and different superscript lower-case letters (a, b) within the same row, are significantly different by the t test (p<0.05). SEM - standard error of the mean.
Treatment of sugarcane silage with urea (conventional or protected) reduced the "a" fraction by 11.8% in relation to the control (mean of 44.07%; Table 3). There was a significant interaction between dose and type of urea, with the highest average observed in sugarcane silage supplemented with 1% conventional urea. The inclusion of 0.5% urea decreased potential degradability of the DM compared with that of the control regardless of urea type. There was no interaction effect for fraction "b" (p>0.05). However, there were effects for both doses, with a higher average at the 0.5% dose and urea type, with a higher mean for protected urea. There was also a difference between the treatment with 0.5% protected urea and the control for fraction "b" (p<0.05). For fraction "c", no difference was observed between treatments, with means of 2.2% per hour (p>0.05).
There was a significant interaction between concentrations and urea types for the potential degradability of dry matter (DM), with the highest average found after 1% urea, regardless of type. The inclusion of urea in the sugarcane silage increased the undegradable fraction of DM by 11.73% in relation to the control silage. There was a significant interaction between dose and urea type, with the highest average observed for 0.5% protected urea.
1Type, effect of urea type; Dose, effect of urea dosage; TxC: interaction effect between type and dose of urea; AxCTL: effect of the additive compared to the control.
* and ** significant at the level of 5 or 1% significance respectively.
Means followed by an asterisk differ from the control by Dunett’s test (p<0.05). Means followed by different superscript upper-case letters (A, B) within the same column, and different superscript lower-case letters (a, b) within the same row are significantly different by the t test (p<0.05). SEM - standard error of the mean.
Discussion
Sugarcane has intrinsic characteristics, such as low buffering power and high soluble-carbohydrate concentration, which allow adequate conservation, even with a DM content below 28%.
Monteiro et al. (2011) suggested that 28% DM is the minimum requirement for proper fermentation since the rapid drop in pH inhibits the development of undesirable microorganisms. The additives did not alter the DM content of the silages, which were all below 28%. The absence of any effect of the additives can be explained by the low proportions of urea added to the silage, and the fact that they are deterioration-inhibiting and not absorbent additives (Castro Neto et al., 2008) (Table 1).
Even with DM values below those recommended for adequate fermentation, the pH values were all less than 4.1, which is sufficient to restrict the development of undesirable microorganisms (Arriola et al., 2011; Ogunade et al., 2017). The low pH values of silage are due to the low buffer capacity of sugarcane, even when a relatively small amount of acid is produced during silage (Pedroso et al., 2014). However, the pH of the silage with 1% conventional urea was higher than the control, although within the limit mentioned above. The high pH observed on the 1% conventional urea treatment was associated with increased ammonia release compared to treatment with the same dose of protected urea. Additionally, the high pH raised the buffering power of the ensiled material, as ammonia absorbs H+ ions present in the medium, neutralizes them, and retards the decrease of the pH (Ferreira et al., 2007) (Table 1).
Treatments with 1% urea showed higher values of N-NH3 compared to 0.5% urea treatments. This increase in N-NH3 content is related to the transformation of urea to ammonia; the higher concentration of urea released higher amounts of N-NH3 in the medium. This result is consistent with the report of Freitas et al. (2017), who observed a linear increase in the percentage of N-NH3 with increasing urea concentrations in millet silage (Table 1).
With regard to urea type, protected urea presented the lowest N-NH3values.This is related to the polymers that cover urea molecules, reducing urease action on urea and controlling ammonia release into the medium. Lopes and Evangelista (2010) reported that ammonia originating from the urease action on urea has an inhibitory effect on yeast growth, thereby reducing DM losses in the silage. Thus, a better evaluation of the N-NH3 level must be made for sugarcane silage, since it can improve silage conservation when it does not exceed a maximum of 10%, according to Costa et al. (2016).
The addition of urea, independently of concentration, reduced gas losses compared to the control treatment. This reduction was associated with the inhibition of breakdown of soluble carbohydrates to release carbon dioxide (Yuan et al., 2017), the main metabolic product related to gas losses. This inhibitory effect is attributed to ammonia production inside the silo, which inhibits fungal and yeast growth (Kung Jr. et al., 2000; Cao et al, 2014.). These microorganisms ferment soluble carbohydrates, producing ethanol, CO2, and volatile compounds that cause DM losses (McDonald et al., 1991). The addition of urea did not influence effluent losses; this is another source of nutrient loss as leaching of soluble compounds can occur, leading to a reduction in the nutritional value of silage (Gebrehanna et al., 2014).
The CP was higher in the silage with 1% urea (15.05%) compared with the silage with 0.5% urea (9.25%), which resulted from the greater addition of non-protein nitrogen in this treatment. Urea is a source of non-protein nitrogen, with 45% N for conventional urea and 39% for protected urea. This inclusion increases the total nitrogen content in silage, which is computed as CP.
Urea type and concentration had variable influences on the fibrous fraction of sugarcane silage compared to the control silage. All the silage treated with urea differed from the control in NDF content, suggesting that the released ammonia resulted in alkaline hydrolysis with partial solubilization of hemicellulose (Lopes and Evangelista, 2010; Carvalho et al., 2012). Another possible mechanism is that the greater preservation of non-fibrous carbohydrates promoted by the additive caused a dilution effect on NDF content (Jacovaci et al., 2017) (Table 2). Only the silage treated with 1% conventional urea showed difference with the control silage; this result may be associated with a higher release of ammonia, causing a more intense alkaline hydrolysis of this fraction.
Lignin content only differed from the control for the silage treated with 1% urea. Van Soest (1994) mentioned that during alkaline treatment, some lignin and silica could be dissolved in the process; so, a higher dose of urea might induce a higher rate of hydrolysis of these constituents.
There was an increase of the EE contents in the silages treated with either 1% conventional or protected urea. A similar result was reported by Silva et al. (2014a), who found an increase in EE content for sugarcane silage after 1% urea treatment. With regard to urea type, an increase in EE values was observed in the silage treated with protected urea. This increase in EE content (Table 2) may be related to the coatings used in protected urea; these are soluble in organic solvents and are counted as lipids. Among the urea encapsulation techniques used by the industry, we can highlight the use of elemental sulfur, polymers, and waxes (Valderrama et al., 2014).
The highest degradability was observed in silage treated with either 1% protected or conventional urea. These treatments were associated with higher ammonia release, providing a higher rate of hemicellulose solubilization. According to Van Soest (1994), alkaline agents, such as urea, act on covalent ester-type bonds between lignin and hemicellulose to increase solubility of the fibrous fraction.
The use of urea in sugarcane silage decreases alcoholic fermentation and soluble carbohydrate losses and promotes quantitative gains due to lower gas losses (Vieira et al., 2017) and qualitative gains due to increased digestibility (Araki et al., 2017). However, in this study, urea reduced the effective degradability by 8.71% compared to silage without the additive. Other studies, such as that by Fortaleza et al. (2012), reported a similar result for sugarcane silage following the addition of 1% of urea. The latter report pointed out that the cause of this effect is unclear. Nevertheless, forage treatment with alkaline substances generally provides an increase in the effective degradability of DM (Rossi Junior and Schogor, 2006). Schmidt et al. (2007) compared sugarcane silage without additives or with 5% urea, showing that the DM in silage with urea had high effective degradability and NDF content was reduced to produce this high effective degradability.
In the present study, the NDF content also decreased in treated silages compared to the control, but there was no improvement in effective DM degradability. Schmidt et al. (2007) reported that qualitative variations of NDF fraction might influence degradability.
Based on this premise, together with technology advances and genetic improvements, new varieties of sugarcane for animal feed have been developed and made available in the market. Thus, variety IAC 86-2480 used in this study presents higher values of ruminal parameters when compared to variety RB72-454 used by Fortaleza et al. (2012). In these two varieties (IAC 86-2480 and RB72-454) no effect of urea inclusion on ensiling was observed with regard to effective degradability. However, a study on another variety at Instituto Agronômico do Paraná found that the addition of 1% urea in sugarcane silage increases effective degradability of DM to 15.36% (Rossi Junior and Schogor, 2006). A study with four sugarcane varieties (RB 855536, RB 765418, SP 80-1842, and SP 80-1816) observed variation in the effective degradability of DM and the readily soluble fraction, because the quality and structural arrangement of the fibers were different (Spínola et al., 2017). This variation in ruminal kinetic behavior using different varieties and additives has also been reported by Silva et al. (2014b).
With respect to ruminants, DM effective degradability of sugarcane silage tends to increase as the amount of ammonia in the silage rises. Ammonia is a source of nitrogen for fibrolytic and cellulolytic bacteria, and increased bacterial activity improves degradation of the fibrous fraction (Van Soest, 1994). This contribution of urea to the increase of nitrogen compounds is supported by the ammoniacal nitrogen and crude protein results. However, in the present study nitrogen increase was not used by ruminal bacteria since degradation rate of the insoluble fraction "b" to fraction "c" was constant, at 2.2% per hour. More studies are needed to elucidate the basis of this effect.
The use of protected or conventional urea at 1% of natural matter at the time of ensiling of sugarcane reduces losses by gases and improves the nutritional composition, with increase of crude protein (CP) content and reduction of fibrous fraction.