Effect of slow-release urea microencapsulated in beeswax and its inclusion in ruminant diets

Small Ruminant Research 179 (2019) 56–63

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Effect of slow-release urea microencapsulated in beeswax and its inclusion in ruminant diets

T

A. de B. Carvalhoa, A.L. da Silvaa, A.M. de A. Silvaa, A.J. Nettoa, T.T.B. de Medeirosa, J.M. Araújo Filhoa, D.L. da S. Agostinib, D.L.V. de Oliveirac, S.E. Mazzettoc, L.R.V. Kotzebuec, ⁎ J.R. Oliveirac, R.L. Oliveirad, , L.R. Bezerraa a

Federal University of Campina Grande, Department of Animal Science, Universitaria Ave., Caixa Postal 61, Patos, Paraíba, Brazil State University of São Paulo, Department of Physics, Chemistry and Biology, Roberto Simonsen Street, 305, 19060900, Presidente Prudente, São Paulo, Brazil c Federal University of Ceara, Department of Organic and Inorganic Chemistry, Contorno Street, 60451970, Fortaleza, Ceará, Brazil d Federal University of Bahia, Department of Animal Science, Adhemar de Barros Ave., 500, Ondina, 40170110, Salvador, Bahia, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Intake Microspheres N-NH3 Nitrogen balance

The objective of this study was to evaluate the effects of including slow-release urea microencapsulated in beeswax in the diet of adult sheep on intake, digestibility, and nitrogen (N) balance. Two microencapsulated system formulations were developed with ratios (wt/wt) of 1:2 (UME1:2) and 1:2 + sulfur (UME1:2+S) between the core (urea) and encapsulant (beeswax). The UME1:2 formulation had greater yield values, microencapsulation efficiency and thermal stability than UME1:2+S. The most efficient formulation (UME1:2) was added to the diet of twenty-four uncastrated male sheep, adult, crossbred Santa Inês, 9 months old with an average body weight of 28.2 kg ± 0.6 kg. The experimental arrangement was a completely randomized design in which the animals were distributed into four treatments that included different levels of urea microencapsulated in beeswax, UME1:2 without S (0, 1.5, 3.0 and 4.5% in total dry matter (DM)), and six replicates in a total experimental period of 19 days, with 14 days for adaptation. The inclusion of urea microencapsulated in beeswax did not affect (P > 0.05) DM, crude protein, ether extract, ash, total carbohydrate or crude energy intake or digestibility. In addition, the nonfibrous carbohydrate (NFC), neutral detergent fiber (NDF), and acid detergent fiber (ADF) intake, N intake, N excretion (urinary and fecal), N absorbed, N retained, N retention/N intake and N retained/N absorbed were not affected (P > 0.05) by the inclusion of urea microencapsulated in beeswax. However, there was a linear increase in NDF (P = 0.031) and ADF (P = 0.004) digestibility as the inclusion level of encapsulated urea increased from 0 to 4.5%. The NFC digestibility showed a quadratic increase (P < 0.001) with the greatest digestibility at 3.0% microencapsulated urea. Beeswax was shown to be an efficient microencapsulant to obtain microparticles containing urea, and the formulation of UME1:2 without the addition of S is recommended since it showed better yield and efficiency than the formulation with S. The inclusion of up to 4.5% urea microencapsulated in the lipid matrix of beeswax is recommended to improve NDF and ADF digestibility without affecting intake and N balance.

1. Introduction Urea is a solid, water-soluble and hygroscopic organic compound that is chemically classified as an amide and belongs to the group of nonprotein nitrogen compounds. Ruminants, through microorganisms present in the rumen, are capable of transforming both the nitrogen derived from true protein and that derived from some nonprotein nitrogen (NPN) compounds, such as urea, ammonium sulfate and biuret, into protein with a high nutritional value (Knaus et al., 2001). Thus, the

⁎

use of urea in the diet of these animals can save nitrogen sources that can be used for humans or other monogastric animals (Holder et al., 2015; Medeiros et al., 2018). The addition of urea to ruminant diets has allowed better utilization of relatively low-quality roughage feeds, such as straw and hay (Cardoso et al., 2018). Therefore, urea can be included in ruminant diets with the main purposes of replacing protein and reducing the cost of the feed or with the objective of raising the nitrogen (N) content of low-quality roughage, increasing its intake and use (Geron et al., 2018).

Corresponding author. E-mail addresses: [email protected], [email protected] (R.L. Oliveira).

https://doi.org/10.1016/j.smallrumres.2019.09.005 Received 20 November 2018; Received in revised form 6 June 2019; Accepted 4 September 2019 Available online 11 September 2019 0921-4488/ © 2019 Elsevier B.V. All rights reserved.

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Brazil, in strict accordance with the recommendations contained in the Guide of the National Council for the Control of Experiences in Animals. The protocol was approved by the Ethics Committee on Animal Experiments (ECAE) with Permit Number 070/2016. However, as urea is a product with a risk of toxicity for sheep, the ECAE requested a reduction in the number of animals used (replications). Thus, twentyfour animals were used, with six sheep (replicates) per treatment.

Research has proven that the N-NH3 peak in the rumen is usually 1 h after feeding when N-NH4 is supplied, while for true protein sources, this peak occurs approximately 3–5 h after feeding (Calomeni et al., 2015; Geron et al., 2016). The considerable amount of ureolytic activity in the rumen, together with the need to adapt animals to diets including urea, has led to the development of products that allow a gradual release of urea in the ruminal environment to increase the production of microbial protein and reduce cases of toxicity (Alves et al., 2014, 2016). In addition, the sulfur (S) content in diets with high levels of N-NH4 is low; thus, the microbial synthesis of sulfur amino acids (methionine, cysteine and cystine) may be hindered by limiting the use of N-NH4, making sulfur supplementation necessary in diets with high NPN levels (Pereira et al., 2008). The main commercial sources of sulfur are ammonium sulfate (24% S) and calcium sulfate (17% S), and the addition of S to diets containing urea can improve protein synthesis in the rumen and improve animal protein performance by increasing the flow of microbial protein and the amino acid supply in the small intestine (Baker et al., 1995). Although considered a relatively new method, microencapsulation has been used since 1970 as a packaging technology involving thin polymeric coverings applied to solid or liquid droplets or gaseous material to form small particles called microcapsules, which may release their content over time or under certain conditions (Todd, 1970). Among the methods for producing microencapsulated systems, lyophilization, also called freeze-drying, is defined by Azeredo (2005) as a technique that uses low temperatures to preserve the core. This technique is based on dehydrating the previously frozen product and then subjecting it to sublimation under vacuum. The microencapsulation process can be carried out by lyophilizing an emulsion of the core material with an encapsulant. The method generates products of excellent quality, since it reduces the changes associated with high temperatures (Medeiros et al., 2018). In this regard, an interesting option with desirable properties as an encapsulating agent for the slow release of urea is animal wax, in particular beeswax, since it meets the requirements for a good coating due to its high hydrophobicity, excellent wet strength, and melting point of 65 °C (Souza et al., 2017). Wax is a lipid substance used mainly in the pharmaceutical and cosmetics industry and is secreted by bees through four pairs of ceriferous glands located in their lower abdomen (Kerr and Amaral, 1960). In beekeeping, wax is considered a byproduct. The production of wax by a hive is variable; however, it is estimated that under good productivity conditions, wax production is approximately 10.8 kg/year (Silva et al., 2003). In addition, beeswax, which solidifies in the form of thin, almost transparent sheets, contain ceric and palmitic acid, hydrocarbon esters, monohydric alcohols, oils, esters, fats, cerolein, vitamin A, and traces of propolis, pollen and different pigments (Barros et al., 2009). In addition, beeswax is an electrical insulator, melts at 63–64 °C, softens beginning at 35 °C, and has a density close to that of water (Fratini et al., 2016). Thus, we hypothesized that the microencapsulation of N-NH4 in beeswax will allow a slow release of the product, reducing the risk of animal toxicity, while the addition of a sulfur source to the system will increase the availability of nitrogen for microbial protein synthesis, stimulate nutrient intake and digestibility, and improve the N balance due to the greater availability of N-NH4. The objective of this research was to evaluate the effects of the slow release of NPN into the rumen via diets including microencapsulated urea with or without sulfur enrichment, using beeswax as the encapsulant and freeze-drying as the drying method, on the intake, digestibility and the N balance.

2.2. Obtaining the microencapsulated systems Two microencapsulated system formulations were developed with ratios (wt/wt) between the core (urea) and encapsulant (beeswax) of 1:2 without a sulfur (S) source (UME1:2) and 1:2 with magnesium sulfate heptahydrate as the S source (UME1:2 + S). The ratio of the nitrogen and sulfur sources was 10:1 (urea/sulfur). The formulations were obtained by emulsification/lyophilization and were differentiated by the presence or absence of a sulfur source, making it possible to determine the influence of sulfur on the rate of incorporation of the nitrogen source. To prepare the emulsions that resulted in the microencapsulated products used in the experiments and for initial characterizations, for each system, urea and the sulfur source (magnesium sulfate heptahydrate) were weighed on an Adventurer AR2140 analytical balance (Mark Ohaus, Parsippany, USA) in separate beakers and dissolved completly in water. The beeswax was then weighed in another beaker and melted in a water bath at 85 °C. Shortly afterward, the actual preparation of the emulsions began. The emulsions were prepared under heat due to the rapid solidification of wax once it was removed from the heating source. Soya lecithin (mass corresponding to 5% of the mass of beeswax) was added to the beaker containing the molten wax and mixed in a mechanical stirrer with the aid of a mixer (Mixer 200, Mondial, Osaco City, São Paulo state, Brazil). Before addition, the urea solution was supplemented with the sulfur solution, and both were heated to the same temperature as the molten wax and then slowly added to the wax, followed by constant stirring at 12,000 × g for 5 min. Finally, the emulsions for the UME2:1 and UME2:1 + S formulations were transferred to labeled plastic containers and frozen in a horizontal freezer at -25 °C for 24 h. After that period, they were dried first in an Alpha 1–4 LD Plus lyophilizer (Brand Christ, Osterode AM Harz, Germany) at a pressure of 0.11 mbar and a temperature of -60 °C for 24 h and then in a circulation oven at 55 °C for 6 h (Medeiros et al., 2018). These parameters were established from previous tests. The microencapsulated product (UME1:2 + S and UME1:2 + S) was processed in a crusher to obtain homogeneous material and subsequently conditioned at room temperature. 2.3. Evaluation of the microencapsulation process 2.3.1. Microencapsulation yield and efficiency The microencapsulation yield (MY) was based on the masses of the urea, sulfur source (when present in the system), beeswax and soy lecithin (emulsifier) used in the preparation of the emulsions and the final mass after drying and was calculated by the following equation: MY = (Mfinal/Minitial) × 100; where Mfinal is the mass of the microencapsulated product after drying, and Minitial is the dry mass of urea, the sulfur source, beeswax and lecithin. The microencapsulation efficiency (ME) was used to evaluate the urea retention capacity of the beeswax matrix and was determined based on the content of urea and urea + sulfur inserted into the microcapsules and the content that remained after the process. The efficiency was calculated by the following equation: ME = (Ureal/ Utheoretical) × 100, where ME = microencapsulation efficiency; Ureal = real content of the retained urea and urea + sulfur; and Utheoretical = the urea and urea + sulfur content that was initially inserted into the capsules.

2. Materials and methods 2.1. Location and ethical considerations The experimental trial was developed at the Federal University of Campina Grande - UFCG at the Center for Rural Health and Technology, 57

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2.3.2. Scanning electron microscopy (SCEM), thermogravimetry (TG), differential scanning calorimetry (DSC) and Fourier transform infrared spectrometry (FTIR) The microstructure of the microencapsulated urea formulations was studied by SCEM. To obtain micrographs of the samples, they were first covered with a thin layer of gold by an SC-701 metallizer (Mark Sanyu electron, Tokyo, Japan). For this process, the samples were fixed with carbon tape, and the metallization time was 3 min under an amperage of 10 mA. The micrographs were obtained on a Vega 3 scanning electron microscope (Mark Tescan, Kohoutovice, Czech Republic) with an acceleration voltage of 30 kV. To obtain micrographs of the beeswax and microencapsulated systems, they were first covered with a thin layer of gold by a Q150R ES (Quorum, Lewes, England) metallizer. For this process, the samples were fixed with carbon tape, and the metallization time was 3 min under an amperage of 10 mA. The micrographs were obtained using an SSX-550 scanning electron microscope (Shimadzu, Kyoto, Japan) with an acceleration voltage of 20 kV. The thermogravimetry (TG) curves were obtained by a TGA/SDTA 851 thermal analyzer (Mettler Toledo Mark) with an inert atmosphere (flow of 50 mL/min), a heating rate of 10 °C/min, and a temperature range between 30 and 600 °C, using platinum crucibles containing approximately 3.0 mg of sample. The DSC curves of the materials were obtained by a DSC-50H (Shimadzu Mark, Kyoto, Japan) under an inert atmosphere (flow of 50 ml / min), a heating rate of 10 °C/min, and a temperature range between 30 and 400 °C, using aluminum hermetic crucibles containing approximately 2.5 mg of sample. Infrared spectra were obtained on an FT-IR/NIR FRONTIER spectrophotometer (Perkin Elmer®, Waltham, USA) using an attenuated total reflectance (ATR) accessory and a zinc selenide (ZnSe) crystal at a resolution of 4 cm−1 with an arithmetic mean of four scans between 500 and 4000 cm−1.

Table 1 Proportion and chemical composition of the ingredients and experimental diets containing different levels of urea microencapsulated (UME2:1) without sulfur in a beeswax matrix. Item

Ingredients

Chemical composition (g/kg DM) Dry matter (g/kg as fed) Ash Crude protein Ether extract Neutral detergent fiber Acid detergent fiber Nonfibrous carbohydrate Crude energy

TiftonSoybean Ground corn UME2:1a 85 hay meal 923 907 889 979 83.3 70.1 15.1 3.20 96.1 489 93.9 939 26.4 54.9 30.1 540 770 198 158 119 431 95.2 43.0 79.9 315 487 751 564 46.2 43.5 48.4 105 Urea microencapsulated in beeswax (UME2:1) 0.0% 1.5% 3.0% 4.5%

Ingredient proportion (g/kg DM) Tifton-85 hay Soybean meal Ground corn Microencapsulated urea Mineral mixtureb Chemical composition (g/kg DM) Dry matter (g/kg as fed) Ash Crude protein Ether extract Neutral detergent fiber Acid detergent fiber Nonfibrous carbohydrate Crude energy

600 108 282 0.00 10.0

600 78.0 297 15.0 10.0

600 48.0 312 30.0 10.0

600 4.00 341 45.0 10.0

912 62.0 137 30.3 528 281 663 46.6

913 60.0 138 37.0 526 280 651 46.0

914 60.0 139 44.1 525 279 640 45.5

914 55.5 134 50.0 522 277 642 44.9

a

UME2:1 = Urea microencapsulated without sulfur in a beeswax matrix. Guaranteed levels Ircafós (for active elements): 120 g calcium, 87 g phosphorus, 147 g sodium, 18 g sulfur, 590 mg copper, 40 mg cobalt, 20 mg chrome, 1800 mg iron, 80 mg iodine, 1300 mg manganese, 15 mg selenium, 3800 mg zinc, 300 mg molybdenum, and maximum 870 mg fluoride. Solubility of phosphorus citric acid:2–95%. b

2.3.3. Moisture and nitrogen (N) content The moisture assay was performed by the gravimetric method in a TE-391/1 (Tecnal) oven with circulation and air renewal at 105 °C until constant mass, while the water activity was verified by a 3TE water activity meter (Marca Aqualab) at 25 °C. The nitrogen assays were conducted according to the Kjeldahl method described by the AOAC (2012), using a TE-0363 (Tecnal) nitrogen distiller.

soybean meal, a mineral premix, and urea microencapsulated in beeswax, and this concentrate was included in the total diet or TMR. The formulated diets were isoproteic (CP mean 137 g/kg DM) and isoenergetic (crude energy-CE mean 46.0 g/kg DM) according to the NRC (2007) guidelines for an average daily gain of 150 g (Table 2).

2.4. Animals, diets and general procedures From the results obtained in the laboratory, urea microencapsulated in carnauba wax from a 1:2 formulation (UME1:2) of beeswax and urea without a sulfur source, corresponding to 33% of the average real value of urea, was chosen for the in vivo experiments. Twenty-four noncastrated male sheep crossbred Santa Inês, 9 months old with an average initial body weight (BW) of 28.2 kg ± 0.6 kg, were randomly assigned to 1 of the 4 treatments (n = 6), which consisted of the inclusion of different levels of urea microencapsulated in carnauba wax (formulation UME1:2) (0%, 1.5%, 3.0% and 4.5% UME2:1 inclusion) based on dry matter (DM). The total experiment period was 19 days, with 14 days for the animals to adapt to the diets and stables and five days for data collection. Prior to the 19 experimental days, all animals were treated for internal and external parasites with ivermectin (Ivomec Gold, Merial®, Salvador, Bahia, Brazil) and vaccinated against clostridiosis (Sintoxan, Merial®, Sao Paulo, Brazil). The sheep were housed in metabolic cages (1.5 by 1.5 m) equipped with water and feed troughs. They received water ad libitum and were fed twice daily (07:00 and 14:00 h) with a total mixed ration (TMR) containing 60% hay (chopped Tifton-85) and 40% concentrate. The feed refusals were collected and weighed daily, and the amount of feed offered was adjusted to allow 10% refusal. Before the experiment, the feed components were separately chemically analyzed (Table 1) in triplicate. The concentrate mixture was composed of ground corn,

2.5. Intake, digestibility and nitrogen (N) balance The nutrient intake was estimated based on the difference between the total amount of each nutrient contained in the feed offered and the amount in the refusals. The digestibility assay was performed on days 14 to 19 of confinement using the total collections of refusals, urine and Table 2 Mean and standard deviation ( ± SD) of the microencapsulation yield and efficiency, total nitrogen and crude protein (CP) contents of urea microencapsulated in beeswax formulations [UME1:2 with sulfur (S) and UME2 (1:2) without S]. Item (%)

UME formulation UME2:1 + S

Microencapsulation yield Microencapsulation efficiency Nitrogen (N) total Crude protein Moisture (%) Water activity (Aw) at 25 °C

90.8 98.5 14.6 91.4 1.72 0.42

± ± ± ± ± ±

0.02 2.70 0.40 2.49 0.18 0.05

UME2:1 b b a a a a

92.5 99.6 14.8 92.4 1.83 0.52

± ± ± ± ± ±

0.01a 3.11 a 0.40 a 2.80 a 0.10 a 0.015 a

Means followed by the same letters do not differ according to Tukey’s test: significant at P ≤ 0.05 and trend at 0.05 ≤ P ≤ 0.1. 58

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Fig. 1. Scanning electron micrographs (SCEM): A) Beeswax (2 μm), B) urea (50 μm) and C) magnesium sulfate (50 μm), D) beeswax formulation UME1:2 without sulfur (S) (1 μm), and E) UME2 (1:2) + S (2 μm).

use of an autoclave. The autoclave temperature was maintained at 110 °C for a period of 40 min. The nonfibrous carbohydrate (NFC) was determined by the equation calculated by Hall (2000): NFC = 100 – [(CP – CP from urea + urea) + NDF + EE + Ash.

feces during this period. Each metabolic cage was equipped with a device for the separation and total collection of feces and the total collection of urine. During the five days of data collection, the feed offered, feed refusals, feces and urine were weighed individually. To avoid the loss of nitrogen compounds from the urine by volatilization, a 10 N hydrochloric acid solution was placed in the container before collection at a volume within 10 ml of the amount of urine produced the previous day. The digestibility coefficients (DCs) of DM, CP, NDF, and ether extract (EE) were calculated using the equation DC = (kg of the portion ingested − kg of the portion excreted)/(kg of the portion ingested) × 100. The TDN intake and TDN concentrations were calculated according to Sniffen et al. (1992) using the equation ITDN = (ICP − CPf) + 2.25 (IEE − EEf) + ITC − TCf), where ITDN, ICP, IEE, and ITC represent the intake of TDN, CP, EE, and total carbohydrates, respectively, and CPf, EEf, and TCf refer to CP, EE, and the total carbohydrate excretion in the feces, respectively. The concentrations of TDN were obtained using the equation TDN (g/kg) = (ITDN/intake of DM) × 100. The nitrogen balance (NB) was calculated from the amount of nitrogen consumed (g/d) and the amounts of nitrogen excreted in the feces and urine. N retention was calculated by the difference between the NB and BEN (basal endogenous nitrogen). The losses of endogenous tissue and dermal N were considered to be 0.35 and 0.018 in metabolic weight, respectively (AFRC, 1993). The BEN was obtained using the equation BEN (g/d) = (0.018 + 0.35) × BW0.75.

2.7. Statistical analysis The first part of the experiment was conducted in a completely randomized design with two treatments [microencapsulated urea in beeswax (UME1:2 + S) with or without sulfur (UME2:1)] with ten replicates per analysis. The characterization results were interpreted statistically through analysis of variance, and the Tukey test was applied to compare the means. The second part of the study used a completely randomized design with four treatments and six replicates per treatment. The PROC GLM procedure in SAS® 9.1 (SAS, 2003) was used. Polynomial contrasts were used to determine the linear and quadratic effects of the amount of microencapsulated urea (UME1:2 without S). P-values less than 0.05 were considered significant, and trends were defined at 0.05 ≤ P ≤ 0.10. 3. Results 3.1. Evaluation and characterization of the microencapsulated systems The two formulation systems tested had relatively high yield values, which were greater (92.5%) for the UME1:2 without S (P ≤ 0.05) than for the UME1:2 + S (90.8%) formulation (Table 1). These values indicate that the microencapsulation of urea in the lipid matrix of beeswax, through lyophilization, is viable for optimal use, demonstrating a low degree of loss of the material during processing. The ME was high in both formulations but beng greater (P > 0.05) for the UME1:2 without S formulation (99.6) than for UME1:2 + S (98.5%). The quantification of the nitrogen and CP contents of the two microencapsulated systems revealed that the two formulations had similar (P > 0.05) concentrations of N (mean = 14.6%) and CP (mean = 91.5%) without the addition of sulfur affecting the process of microencapsulation. Regarding the moisture content, the formulations UME1:2 and UME1:2 + S had similar (P > 0.05) values of 1.83 and

2.6. Chemical analysis Samples of the ingredients, refusals, and feces were predried in a forced-air ventilation oven at 55 °C for 72 h. Then, samples were ground in a Wiley knife mill with a sieve size of 1 mm. The samples were stored in plastic jars with lids, labeled, and subsequently analyzed to determine the DM (method 967.03), ash (method 942.05), CP (method 981.10), and ether extract (EE; method 920.29) contents (AOAC, 2012). Analyses for the determination of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were performed according to Van Soest et al. (1991) with the changes proposed by Senger et al. (2008) to include the 59

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Fig. 2. A) Thermogravimetric (TG), B) differential scanning calorimetry (DSC) curves and C) Fourier transform infrared (FTIR) spectrograms for urea (U), beeswax (BW), and both formulations of urea microencapsulated in beeswax [UME1:2 with sulfur (S) and UME2 (1:2) without S].

at 2915 and 2847 cm−1, which were attributed to the stretching of the C–H bond, and at 1736 cm−1, which was attributed to the stretching of the C]O bond. The sulfur source used had a characteristic band at 1110 cm−1, which was attributed to the SO42- ion, since magnesium sulfate heptahydrate was used in the study. According to the results observed in relation to the formulations, the UME1:2 without S formulation exhibited improved yield, ME and thermal stability, making the urea more effectively protected and therefore more suitable for gradual nitrogen release. For this reason, this microencapsulated system was used in the in vivo digestibility assay.

1.72%. Another important parameter in the characterization of a powder product is its water activity. The microencapsulated systems UME1:2 and UME1:2 + S showed water activity (Aw) at 25 °C of 0.52 and 0.42, respectively. The microencapsulated systems and their components were subjected to SCEM to verify their morphology, and the beeswax surface (Fig. 1A) was observed to be irregular, full, sealed, poreless and smooth; however, the urea (Fig. 1B) and magnesium sulfate (Fig. 1C) had a rough surface with cracks and pores. The sulfur-free UME1:2 formulation (Fig. 1D) showed a surface quite similar to that of beeswax, indicating that urea was found inside the microparticle, although there was a small amount of urea exposed. In the UME1:2 + S formulation (Fig. 1E), the microstructure was very rough with agglomerates, which indicates the presence of a considerable amount of surface urea, probably due to the presence of the sulfur source, which reduced the effectiveness of the protection and caused the nitrogen source to be exposed. The thermogravimetric (TG) curve of beeswax showed an initial degradation temperature of 294 °C, while that of urea showed an initial degradation temperature of 170.5 °C(Fig. 2A). The DSC curves of all materials showed only endothermic events within the temperature range investigated (Fig. 2B). These events were attributed to the fusion of beeswax (Event 1) and urea (Event 2). The main absorption bands typical of urea appeared at 3428 and 3330 cm−1, which were attributed to the asymmetric and symmetrical vibration of the NeH bond, and at 1672 cm−1, which was attributed to the C]O bond (Fig. 2C). The beeswax showed typical absorption bands

3.2. Intake, digestibility and nitrogen (N) balance The inclusion of urea microencapsulated in beeswax (UME1:2 without sulfur formulation) did not affect (P > 0.05) the intakes (Table 3) of DM, CP, EE, NDF, ADF, ash, total carbohydrates, NFC and crude energy. There was no effect (P > 0.05) of the urea microencapsulated in beeswax on the digestibility coefficients of DM, CP, EE, ash, total carbohydrates, crude energy and TDN. However, there was a linear increase in the NDF (P = 0.031) and ADF (P = 0.004) digestibility with the inclusion of urea microencapsulated in beeswax in the sheep diet. The NFC digestibility showed a quadratic increase (P < 0.001) with the greatest digestibility achieved with the inclusion of 3.0% urea microencapsulated in beeswax. Nitrogen intake (g/day; g/kg0.75), N excretion (g/day) (urinary and 60

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Table 3 Intake, digestibility and nitrogen (N) balance in sheep fed diets with added urea microencapsulated (UME2) in beeswax formulation (1:2) for slow release. Item

Nutrient intake (g/day) Nº animals Dry matter Crude protein Ether extract Neutral detergent fiber Acid detergent fiber Ash Total carbohydrates Nonfibrous carbohydrates Crude energy Digestibility (%) Dry matter Crude protein Ether extract Neutral detergent fiber Acid detergent fiber Ash Total carbohydrates Nonfibrous carbohydrates Crude energy Total digestible nutrients Nitrogen intake Fecal nitrogen excretion Urine nitrogen excretion Nitrogen absorbed N retain N retain /N intake N retain /N absorbed Nitrogen balance (g/kg0.75) Nitrogen intake Nitrogen absorbed N retain a b

SEMa

Microencapsulated urea (%) 0.0

1.5

3.0

4.5

6 785 107 23.8 414 221 48.6 605 191 3656

6 788 108 29.1 415 221 47.3 603 188 3623

6 905 126 39.9 475 253 54.3 685 210 4116

6 813 109 40.6 424 225 45.1 618 193 3648

58.6 64.9 38.0 49.5 30.5 29.1 62.4 90.4 41.8 59.6 Nitrogen balance (g/day) 18.0 4.79 6.80 13.24 6.44 0.32 0.42

60.2 59.8 34.3 57.0 31.5 34.9 68.3 93.2 37.5 63.4

66.1 64.2 46.7 59.2 41.0 39.1 69.7 93.3 38.1 66.3

18.4 5.45 7.27 12.94 5.66 0.25 0.36

1.25 0.93 0.44

1.40 0.98 0.41

P-valueb Linear

Quadratic

– 3.19 4.32 1.06 1.67 8.90 1.87 2.43 7.55 1.45

– 0.87 0.89 0.22 0.89 0.89 0.96 0.90 0.92 0.93

– 0.97 0.96 0.48 0.98 0.98 0.97 0.98 0.99 0.98

64.3 65.1 27.8 62.2 41.4 34.8 68.7 82.8 42.9 64.1

3.43 3.18 4.12 7.56 4.25 5.11 3.56 2.96 1.03 3.56

0.16 0.77 0.63 0.031 0.004 0.26 0.12 0.057 0.16 0.77

0.35 0.70 0.59 0.095 0.024 0.26 0.14 < 0.001 0.35 0.70

21.3 4.69 6.53 16.59 10.1 0.47 0.60

19.6 5.67 6.83 13.94 7.10 0.35 0.48

5.94 2.16 1.56 3.91 2.56 0.05 0.08

0.78 0.85 0.93 0.75 0.60 0.72 0.35

0.95 0.98 0.99 0.92 0.81 0.92 0.64

1.49 1.17 0.71

1.34 0.96 0.48

0.29 0.19 0.14

0.79 0.76 0.57

0.86 0.78 0.71

Standard error of the mean. Significance at P ≤ 0.05 and trend at 0.05 ≤ P ≤ 0.1.

beeswax and has already been tested as a microencapsulating agent for urea, showing high yields for this alternative source of N (Medeiros et al., 2018; Netto, 2018). The urea in the microencapsulated systems showed greater thermal stability than isolated urea, with a higher thermal stability of the UME1:2 system, supporting the results from the micrographs that showed surface urea in the UME1:2 + S system, revealing decreased protection (Fig. 2A). Another widely used slow-release urea formulation is starea (a supposedly slow-release NPN source). The greatest advantage of starea is the gradual release of N in the rumen (Oliveira et al., 2004). However, starea in ground or pellet form presents disadvantages, namely, reduced solubility of the N and segregation in the mixtures, making its use difficult (Pires et al., 2004). The presence of beeswax allowed a tendency for the cassava starch film (used to preserve plants after harvesting) to show improved thermal stability, which was similar to results reported by Jafari et al. (2015); Rocca-Smith et al. (2016), and Zhu et al. (2017). In addition to the higher initial degradation temperature, the UME1:2 system caused a slower mass loss, requiring higher temperatures than urea alone or the UME1:2 + S formulation, with losses of 10, 20 and 30%, respectively. The formulations showed similar values for the loss of 50% mass. This result proves that the system without sulfur has little superficial urea and provides better protection of the urea. The beeswax and urea had melting points of 65.61 and 135.83 °C, respectively (Fig. 2C), which are in agreement with the literature; according to Barros et al. (2016), the ideal melting point for unadulterated beeswax is approximately 64 °C, whereas according to

fecal), N absorbed, N retained, and the N retained/N intake and N retained/N absorbed ratios were not affected (P > 0.05) by the inclusion of urea microencapsulated in beeswax.

4. Discussion 4.1. Evaluation and characterization of the microencapsulated systems The high yield of the microencapsulation of pure urea or sulfur urea demonstrated that the lyophilization technique is suitable for microencapsulating urea in beeswax with little loss of the material during processing. Although excellent yields were obtained with little difference between the formulations, losses still occurred during the transfer of the samples from the emulsion to lyophilization containers by virtue of the viscosity of the sample and its solidification at room temperature. These results were, however, better than those from the lyophilization method used by Gonsalves et al. (2009), where the essential oil of Citrus sinensis (L) Osbeck was encapsulated; chitosan was used as the wallforming polymer and had an 82.8% yield, which was considered optimal by the authors. These results confirm the suitability of lyophilization as a microencapsulation technique for the components (core and encapsulant) of the present study. The nitrogen concentration determination allowed us to estimate the equivalent CP content to determine the actual percentage of urea in the formulations and thereby investigate the efficiency of the process. The high efficiency of the microencapsulation in the two systems demonstrates the retention ability of beeswax as an encapsulant for the core studied. Carnauba wax has characteristics similar to those of 61

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15% DM (Benedeti et al., 2014; Ítavo et al., 2017). Thus, the amount of soybean meal in the diets used in this experiment was reduced, since supplementation with encapsulated urea requires the inclusion of protein sources with low solubility and low ruminal degradation to avoid excess N- NH3 (Broderick and Reynal, 2009). This objective was reached because the treatments did not affect the N balance variables. This result means that although we increased the NPN in the sheep diet by 1.5%, no N was wasted, which could lead to a loss of energy during rumen fermentation. The slow release of N-NH4 by its microencapsulation in beeswax allowed better digestibility of the fibrous (NDF and ADF) and nonfibrous (NFC) fractions of the feed because adequate proportions of rapidly fermentable and moderately fermentable carbohydrates maximize the use of N-NH4, which in turn increases dietary fiber digestibility by increasing the population of ruminal microorganisms (Van Soest, 1994; Sampaio et al., 2009). The use of encapsulated urea has the potential to stimulate protein synthesis, increase fiber degradability, and consequently increase the feed passage rate, promoting DM consumption because the rumen empties faster (Oliveira et al., 2004; Sampaio et al., 2009). Some modified forms of N-NH4 have been developed to obtain slower release of N-NH3, allowing the delivery of larger quantities of urea without causing an ammonia overload or a decrease in food consumption. Examples of such forms are "dehy 100," which is a pelletized alfalfa blend with 32% N-NH4, dicalcium phosphate, sodium sulfate and sodium propionate; "amirea," which is a combination of cereal grains and N-NH4 obtained through a process of extrusion and baking with heat, moisture and pressure causing gelatinization of the starch; and biuret, which originates from the conjugation of two molecules of NNH4 by condensation, has a much slower rate of N-NH3 release and requires a longer adaptation period than urea (Salman, 2008). However, for all these compounds, the tested product can only be used under limited conditions. In the tested beeswax-microencapsulated systems (UME1:2 without sulfur (S) and UME1:2 + S), a high retention of N-NH4 was observed, as well as excellent yield values, confirming that the use of beeswax and the lyophilization technique were appropriate for the purpose of the present study. Compared to the formulation with sulfur, the UME1:2 formulation without added sulfur in the encapsulation process exhibited a higher yield, a higher efficiency of microencapsulation, greater thermal stability, and consequently better protection of the urea, emphasizing the negative influence of the incorporation of a sulfur source in the microencapsulated system. It is worth noting that the sulfur content is normally low in rations with high levels of N-NH4, especially in high-grain diets or diets based on silages from grain processing plants. Therefore, external supplementation outside the encapsulation is recommended to guarantee the microbial synthesis of sulfur amino acids (methionine, cysteine and cystine).

Malavolta (1981), the melting point of urea is approximately 132 °C. The results show that the melting point of the microencapsulated systems did not change from that of beeswax and that it is safe to store or use them in processes involving temperatures up to approximately 65 °C. In the temperature range at which urea melts, an increase in the melting point of the UME1:2 formulation was observed compared to that of free urea. This result indicates that, even if already fused, the wax was able to protect urea but was not as effective in the UME1:2 + S formulation due to the higher exposure of urea, as previously shown by the SCEM and TG results. The formulations had low moisture contents (mean 1.8%) and were considered satisfactory for product conservation. Foods with high residual moisture will deteriorate faster than foods with low residual moisture, and studies show that a moisture content of 4% or less is required for powdered foods (Zayed and Roos, 2004). In addition, the microencapsulation systems showed water activity values ranging from 0.42 to 0.52 for the UME1:2 + S and UME1:2 systems without sulfur, respectively. According to Troller (1980), values below 0.6 for water activity are considered safe from microbial growth. Considering this information, the two systems had values appropriate for food production. The absorption spectra (Fig. 2C) of the microencapsulated systems showed that all the main bands attributed to the isolated urea and beeswax were maintained, attesting that the integrity of each phase was maintained. Because of the lack of reactivity, the wax is suitable to encapsulate urea because the two are chemically compatible. The bands located at 3429 and 3332 cm−1 were the only ones that showed considerable variation in the formulations compared to those of the isolated urea, since there was a displacement to a lower region of the spectrum in addition to a small enlargement. This result indicates a possible interaction of the group in question (NeH) with hydrogen, which is acceptable since urea forms intermolecular interactions (H-bridge). This interaction does not entail any problem for the study. The infrared spectrum of the UME1:2+S formulation showed a band at 1110 cm−1, which was attributed to the sulfate ion, confirming its presence in the system. This band was not observed in the UME1:2 formulation, as expected. 4.2. Intake, digestibility and nitrogen (N) balance The inclusion levels of the microencapsulated urea in beeswax were equivalent to 0.5%, 1% and 1.5% of urea in the total diet, respectively, and since two parts of the microencapsulated system were beeswax (UME1:2), approximately 33% of the microcapsules consisted of urea. In progressive adaptation of the animals to the NPN source, urea supplementation should not exceed 1.0% of the total DM of the diet (Van Soest, 1994; Santos et al., 2017). In the present study, urea was offered without prior adaptation at 1.5% of urea in the total diet for the sheep, and the sheep did not show clinical signs of ruminal alkalosis, which proves the slow release of the urea. In addition, the inclusion of microencapsulated urea in beeswax at a level of up to 4.5% did not change the feed intake of the animals; however, when urea sources are provided in ruminant diets, intake is generally decreased. Most likely, the microbial protein and the NPN supplied by the beeswax-microencapsulated urea were able to meet the animal metabolizable protein requirements (NRC, 2007), since all diets provided a minimum of 12% rumen degradable protein (RDP) in relation to crude energy, as recommended by Silva et al. (2017). Another factor that should be considered is that the treatments had a minimum of 130 g/kg CP as a DM basis, which was able to maintain a balance between CP and crude energy. The amount of protein in the diet affects the conversion of NPN into microbial protein. High protein contents are able to reduce the amount of ammonia used by rumen microorganisms (Azevedo et al., 2015). In diets with adequate levels of RDP, the maximum level of crude protein at which the addition of NPN reduces the use of ammonia for protein synthesis is between 14 and

5. Conclusion Beeswax was shown to be an efficient microencapsulant for microparticles containing urea because it inhibits the hygroscopicity of urea, preventing its solidification during storage and therefore facilitating its homogenization with the other ingredients in the diet. Compared to the formulation with sulfur, the formulation of UME1:2 without added sulfur is recommended since it had a better yield and higher urea content, providing a higher supply of slow-release NPN to the animal. The replacement of conventional soybean protein with up to 4.5% NNH4 encapsulated in the lipid matrix of beeswax increased NDF and ADF digestibility for the diets studied. In addition, the slow release of microencapsulated urea in the rumen reduces the risks of toxicity and ruminal alkalosis.

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