Animal Feed Science and Technology 216 (2016) 68–80 Contents lists available at ScienceDirect Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci Impact of days post-burning and lime as an additive to reduce fermentative losses of burned sugarcane silages Anna Paula T.P. Rotha,∗, Gustavo R. Siqueiraa,b, Carlos H.S. Rabeloa, Carla J. Härtera, Fernanda C. Bassoa, Telma T. Berchielli a, Ricardo A. Reisa a UNESP, Univ. Estadual Paulista, Department of Animal Sciences, Jaboticabal, 14884-900 SP, Brazil b Agência Paulista de Tecnologia dos Agronegócios, Alta Mogiana, Colina, 14770-000 SP, Brazil a r t i c l e i n f o Article history: Received 4 August 2015 Received in revised form 8 March 2016 Accepted 9 March 2016 Keywords: Alkaline treatment Burned silage In natura silage Yeast population a b s t r a c t The objectives of our study were: 1) to investigate the effects of burning and the time elapsed between burning and ensiling on characteristics of sugarcane silages, and 2) to evaluate the effects of lime on fermentation and aerobic stability of in natura and burned sugarcane silages. In trial I, silages were prepared from burned sugarcane that remained in the field for varying number of days post-burning (1, 5, 10, 15, 20, 25, and 30 d). In trial II, the characteristics of burned and in natura sugarcane silages treated with various concentrations of lime (0, 5, 10, 15, and 20 g/kg of sugarcane, on an as-is fresh matter basis) were compared. In trial I, 10-d post-burning, sugarcane crop displayed great degrees brix (18.3◦Bx) and sucrose (677.3 g/kg of sugarcane broth) values. The yeast population in sugarcane crop 1-d post-burning (4.47 cfu/g of fresh forage) was lesser than that 25-d post- burning (7.11 cfu/g of fresh forage). After the silos were opened, all silages showed low pH. The silage from 1-d post-burning had the least dry matter (DM) recovery (637.5 g/kg of DM). The greatest DM recovery was found in the silage prepared 15-d post-burning (740.0 g/kg). Silage from 20-d post-burning displayed the greatest aerobic stability (36.7 h); however, in general, all silages had low aerobic stability (<40 h). In trial II, both the in natura and burned silages had reduced fiber content due to lime addition. Considering the overall mean, burned silages produced 47 g acetic acid/kg of DM against 25.6 g/kg of DM in in natura silages. Lime was more effective in increasing the production of acetic acid in in natura silages, but only when applied at great concentrations (15 and 20 g/kg). DM recovery of in natura silages decreased with increased addition of lime, whereas the opposite effect was observed for burned silages. In natura and burned silages treated with lime at 15 and 20 g/kg had greater aerobic stability (>8 d) than those treated with lesser quantities of lime. Considering the approach in which this study was carried out, a period of 10–15 days is ideal for ensilage of burned sugarcane prior to the silage quality significantly drops. Lime may be used as an additive for both in natura and burned silages since in greater levels (15 and 20 g/kg). © 2016 Elsevier B.V. All rights reserved. ∗ Corresponding author. E-mail address: annapaularoth@yahoo.com.br (A.P.T.P. Roth). http://dx.doi.org/10.1016/j.anifeedsci.2016.03.010 0377-8401/© 2016 Elsevier B.V. All rights reserved. dx.doi.org/10.1016/j.anifeedsci.2016.03.010 http://www.sciencedirect.com/science/journal/03778401 http://www.elsevier.com/locate/anifeedsci http://crossmark.crossref.org/dialog/?doi=10.1016/j.anifeedsci.2016.03.010&domain=pdf mailto:annapaularoth@yahoo.com.br dx.doi.org/10.1016/j.anifeedsci.2016.03.010 1 c ( S t a s s ( b ( ( s f ( c 2 2 2 fi c C w 2 c r m o t s r 2 ( w c w c 2 a A.P.T.P. Roth et al. / Animal Feed Science and Technology 216 (2016) 68–80 69 . Introduction Sugarcane crops display great herbage production (25–40 t/ha of dry matter [DM]) and great energy values at maturity ompared to other crops (Ávila et al., 2009); it is widely used as silage in diets for beef cattle and dairy cows in Brazil Bernardes and Rêgo, 2014; Millen et al., 2009) because the difficult to perform daily cuts of fresh sugarcane on the field. ugarcane is often harvested during the dry season. In this period, there is a great risk of accidental burning, which prevents he use of sugarcane in a direct cut system. Therefore, ensiling may be an effective strategy to avoid greater losses of quality nd DM in this situation, but the maximum time that sugarcane can remain in the field post-burning before quality declines ignificantly is not conclusive (Roth et al., 2010). Conversely, sugarcane ensiling may result in large DM losses (>300 g/kg) during fermentation due to the conversion of ucrose to ethanol by yeast, leading to the reduction in silage nutritive value, aerobic stability, and feed intake by livestock Kung and Stanley, 1982; Pedroso et al., 2005). Thus, the addition, of an alkaline agent (e.g., lime) at the farm level has een recommended for reducing the fungi population and increasing the growth of the lactic-acid bacteria (LAB) population Cavali et al., 2010), as well as to increase DM recovery (Rabelo et al., 2014), and improve the sugarcane silage digestibility Siqueira et al., 2007, 2011a). These treatment effects are relatively well known for in natura sugarcane silage, but few tudies have evaluated the process of ensiling burned sugarcane or the effect of chemical additives on this process. In that ew studies, lime addition increased the in vitro digestibility (Roth et al., 2010) and DM recovery of burned sugarcane silages Siqueira et al., 2011a). Therefore, our objectives were to investigate the effects of the time between burning and ensiling on sugarcane silage haracteristics and the effect of lime on fermentation and aerobic stability of in natura and burned sugarcane silages. . Material and methods .1. Ensiling process .1.1. Trial I In trial I, we used a crop of the IAC 86-2480 sugarcane cultivar after 12 months of growth (third cut). The sugarcane eld was burned at sunset on the day before cutting. The stems were not severed from their roots until harvest taking into onsideration any incidence of accidental fire. On days 1, 5, 10, 15, 20, 25, and 30 post-burning, sugarcane was mechanically harvested using an ensilager (Menta Mit, olhiFlex model, Cajuru, SP, Brazil). Mini-silos (plastic buckets of 7 L capacity) were used in quadruplicate for ensiling, which ere closed with plastic lids and sealed with adhesive tape; a density of 676 kg fresh matter/m3 was obtained. .1.2. Trial II The sugarcane used in trial II was similar to that used in trial I. The sugarcane field was burned at sunset the day before utting, and 1-d post-burning sugarcane was used to ensilage. After harvest, the burned and in natura sugarcane either emained untreated or was treated with lime (micro-pulverized calcium oxide) at 5, 10, 15, and 20 g/kg (on an as-is fresh atter basis). Lime was diluted in water at a ratio of 1.0 kg lime per 4 L of water and applied at 20, 40, 60, and 80 mL/kg f fresh sugarcane to obtain the concentrations described earlier. Lime was then well-mixed with the sugarcane multiple imes to ensure good homogenization. Mini-silos (plastic buckets of 20 L capacity) were used in quadruplicate for ensiling, and were closed with a plastic lid and ealed using adhesive tape; a density of 872 and 562 kg fresh matter/m3 was obtained for burned and in natura sugarcane, espectively. .2. Fermentative losses In both trials, the mini-silos were weighed immediately upon ensiling and after opening at the end of the ensiling period 56 d) to determine gas and DM losses, which were calculated as: Gaslosses(g/kgofDM) = (((Fen × DMen) − (Fop × DMop))/(Fen × DMen)) × 100 (1) here Fop and DMop = forage mass and DM content of the silos at opening, respectively; Fen and DMen = forage mass and DM ontent at ensiling, respectively. DMrecovery(g/kg) = (Fop × DMop)/(Fen × DMen)× 100 (2) here Fop and DMop = forage mass and DM content of the silos at opening, respectively; Fen and DMen = forage mass and DM ontent at ensiling, respectively. .3. Aerobic stability To determine aerobic stability, a silage sample from each mini-silo was placed in a plastic bucket of 7 L capacity and kept t ambient temperature. A data logger was placed in the silage and the silage temperature was measured every hour for 9 70 A.P.T.P. Roth et al. / Animal Feed Science and Technology 216 (2016) 68–80 d. The ambient temperature was measured using data loggers placed close to the mini-silos. Aerobic stability was defined as the number of hours that the silage temperature remains stable before increasing more than 2 ◦C above the ambient temperature (Moran et al., 1996). Additionally, the sum of accumulated daily temperatures was calculated as the sum of the difference between the silage and ambient temperatures after 5 and 9 d (SUM 5 and SUM 9) of aerobic exposure (O’Kiely, 1999). The heating rate was calculated as the maximum recorded temperature divided by the time required reaching the maximum temperature (Ruppel et al., 1995). The pH values were recorded at the initial time (day 0), and after 3, 6, and 9 d of aerobic exposure. 2.4. In vitro gas production and digestibility Animal care and handling procedures used in this study were approved by the Sao Paulo State University’s Animal Care Committee, in agreement with the guidelines of the Brazilian National Council for the Control of Animal Experimentation (CONCEA). Dried silage samples (0.2 g) were placed in 115-mL serum bottles and incubated in a water bath at 39 ◦C (Mauricio et al., 1999) with a rumen inoculum and buffer solution in a ratio of 4:1 (30 mL for each bottle) for 144 h. Prior to incubation, CO2 was continuously purged for 30 min, and the reducing solution was added to the ruminal fluid. The bottles (4 for each treatment) were then sealed with a rubber stopper plus an aluminum crimp cap and stored in a water bath (39 ◦C). For the buffer solution, we used the Kansas State “synthetic saliva” (Marten and Barnes, 1979), which was adapted from the two-stage technique of Tilley and Terry (1963). The ruminal fluid was collected from 2 rumen-cannulated sheep in the morning before feeding. The animals were fed 600 g/kg sugarcane silage treated with lime at 10 g/kg and 400 g/kg concentrate composed of ground corn, soybean meal and urea, on a DM basis. The rumen fluid was filtered through 4 layers of cheesecloth into pre-warmed thermos flasks, homogenized, and mixed with the buffer solution. The accumulated headspace gas pressure was measured using a needle attached to a pressure transducer connected to a visual display (Datalogger pressure- pressDATA 800, MPL, Piracicaba, SP, Brazil). Readings were taken at regular intervals throughout the incubation period and at an increased frequency during the initial lag and rapid fermentation phases (i.e., 2, 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36, 48, 52, 56, 60, 72, 78, 84, 96, 108, 120, 132, and 144 h). To correct changes in the atmospheric pressure, two flasks were incubated without samples (blank) (Pell and Schofield, 1993). The volume of gas in the blanks was subtracted from the reading of gas volume to obtain the true sample gas volume. Since in vitro gas production is mostly due to digestible carbohydrate content, rather than that of protein and fat, we used a multiple regression analysis (Menke and Steingass, 1988) to calculate the digestible organic matter (IVOMD): IVOMD(g/kg) = 14.88 + (0.889 × gas24) + (0.045 × CP) + (0.065 × Ash) (3) where gas24 = gas production after 24 h (mL/0.2 g of DM) and crude protein (CP) and ash contents are expressed as g/kg of DM. 2.5. Chemical analyses One sample of forage and silage from each mini-silo was used for the chemical analyses. Each sample was divided into three sub-samples. The first sub-sample was weighed and placed in a forced air chamber at 55 ◦C for 72 h. After this period, these sub-samples were again weighed, ground in a knife grinder until the particle size reduced to less than 1 mm, and stored in plastic pots for determination of DM (105 ◦C for 12 h) and ash (500 ◦C for 5 h). CP was determined following the methodology recommended by AOAC (1996), method no. 960.52). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined by the sequential method (Robertson and Van Soest, 1981). The NDF was assayed without a heat stable amylase, and NDF and ADF were expressed inclusive of residual ash. Cellulose was measured after hydrolysis in ADF residues in 72% H2SO4 (Van Soest, 1994), and lignin (sa) content was calculated as the difference between ADF and cellulose. The second sub-sample was used to prepare a water extract (Kung et al., 1984) and pH was measured using an elec- trode (model MA522, Marconi Laboratory Equipment, Piracicaba, Sao Paulo, Brazil). Acetic acid was measured using a gas chromatograph (Shimadzu model GC2014, Shimadzu Corp., Kyoto, Japan) equipped with a HP-INNOWax capillary column (30 m × 0.32 mm; Agilent Technologies, Colorado, USA) at an initial temperature of 80 ◦C for 3 min followed by a heating rate of 20 ◦C/min until a final temperature of 240 ◦C was achieved. The amount of total soluble solids (Brix), sucrose content, redox sugars (glucose + fructose), and purity of sugarcane broth also were measured (Bovi and Serra, 1999). The third sub-sample was used for microbiological analyses performed according to Kurtman and Fell (1998). Briefly, a 25 g silage sample from each replicate was homogenized in 225 mL of saline solution (0.85% NaCl) for 1 min. Then 1 mL of this solution was transferred into tubes with 9 mL of saline solution, from which 0.1-mL samples were transferred to Petri plates at dilutions from 10−1 to 10−5. Potato dextrose agar (PDA) was used as a medium to cultivate yeasts. The plates with PDA were kept at 28 ◦C for 72 h. Microbiological data were log-transformed for statistical analysis. 2.6. Statistical analyses Both experiments were performed using a completely randomized design with four replicates. Data from fermentation and aerobic stability were analyzed with a mixed model using the MIXED procedure of SAS (v 9.4, SAS Inst. Inc., Cary), A.P.T.P. Roth et al. / Animal Feed Science and Technology 216 (2016) 68–80 71 Table 1 Chemical composition (g/kg of DM, unless otherwise stated) of sugarcane at different days post-burning and before ensiling. Itema Days post burning SEM P-value Contrastb 1 5 10 15 20 25 30 DM, g/kg as fed 270.4 268.4 241.2 256.7 242.9 242.0 255.3 3.94 < 0.0001Q** Ash 15.3 20.3 14.3 16.3 33.1 28.4 16.0 1.16 <0.0001 C** CP 29.9 29.0 27.3 28.8 30.0 29.0 30.6 0.50 0.0031 Q* NDF 413.7 430.0 405.6 433.8 491.8 464.2 469.2 8.41 <0.0001 Q* ADF 248.7 262.5 255.4 272.3 318.3 294.9 294.5 7.31 <0.0001 Q* Lignin (sa) 38.5 40.5 38.5 44.7 78.3 53.3 47.4 3.39 <0.0001 Q** IVOMD 545.7 539.1 540.1 554.1 555.7 538.9 559.6 8.48 0.4371 NS Brix, ◦ 17.1 18.2 18.3 16.8 17.1 18.1 17.9 0.11 <0.0001 C** Sucrose, g/kg of sugarcane broth 602.2 661.7 677.3 565.7 570.8 562.4 549.4 6.41 <0.0001 C** Pooled sugars, g/kg of sugarcane broth 147.7 161.6 165.2 139.2 140.1 137.4 134.4 1.54 <0.0001 C** Redox sugar, g/kg of sugarcane broth 6.8 5.9 5.7 8.0 8.3 10.4 10.6 0.29 <0.0001 C** Purity, g/kg of total sugars 863.0 888.4 894.8 826.2 821.2 758.7 751.4 8.33 <0.0001 C** Yeasts, log cfu/g 4.47 5.20 5.36 5.28 5.79 7.11 6.14 0.09 <0.0001 C* a DM = dry matter; CP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber; IVOMD = in vitro organic matter digestibility. b NS = no significant; Q = quadratic effect; C = cubic effect. * f a w w S m o p t s t o p w t w S b k P < 0.05. ** P < 0.01. actoring in the number of days that sugarcane remained in the field post-burning (trial I), silage type (in natura or burned) nd lime levels (trial II) as fixed effects, and the residual error as a random effect. The following general models were used: Trial I Yij = � + Di + eij here Yij = response variable; � = overall mean; D = effect of ensiling in different post-burning days i; and eij = error term. Trial II Yijk = � + Si + Lj + SLij + eijk here Yijk = response variable; � = overall mean; S = effect of silage type (in natura or burned) i; L = effect of lime levels j; L = effect of the interaction between silage type i and lime levels j; and eijk = error term. Differences between means were determined using the DIFF option of the LSMEANS statement, which differentiates eans based on Fisher’s F-protected least significant difference test. Contrasts were constructed, and the single degree- f-freedom orthogonal comparisons included the linear, quadratic, and cubic effects of ensiling on different numbers of ost-burning days (trial I) and lime levels (trial II). For both trials, quartic effects were studied; however, we did not use his effect even when was it significant because a biological explanation would be difficult and likely would not make much ense. When there was significant interaction between the factors studied in trial II, contrasts were constructed based on hat interaction. Differences were declared significant at P ≤ 0.05. The pH values over aerobic exposure time were analyzed as a completely randomized design with repeated measures ver time. The covariance matrix that best fit the data according to the Bayesian information criterion (BIC) was selected to erform the analysis. The following general models were used: Trial I Yijk = � + Di + Tj + DTij + eijk here Yijk = response variable; � = overall mean; D = effect of ensiling on post-burning day i; T = effect of aerobic exposure ime j; DT = effect of interaction between ensiling on post-burning day i and aerobic exposure time j; and eijk = error term. Trial II Yijkl = � + Si + Lj + SLij + Tk + STik + LTjk + SLTijk + eijkl here Yijkl = response variable; � = overall mean; S = effect of silage type (in natura or burned) i; L = effect of lime levels j; L = effect of interaction between silage type i and lime levels j; T = effect of aerobic exposure time k; ST = effect of interaction etween silage type i and aerobic exposure time k; LT = effect of interaction between lime levels j and aerobic exposure time ; SLT = effect of interaction among silage type i, lime levels j and aerobic exposure time k; and eijkl = error term. 72 A.P.T.P. Roth et al. / Animal Feed Science and Technology 216 (2016) 68–80 Table 2 Chemical composition, fermentation, and aerobic stability of sugarcane silages prepared 1–20 days post burning. Itema Days post burning SEM P-value Contrastb 1 5 10 15 20 25 30 Chemical composition, g/kg of DM DM, g/kg as fed 162.5 168.5 165.2 179.7 168.7 180.8 171.2 2.50 0.0008 Q* Ash 25.3 24.2 21.3 20.5 35.4 32.0 24.4 1.29 <0.0001 C** CP 45.2 40.4 41.8 41.1 44.7 42.9 49.2 0.77 <0.0001 Q** NDF 704.4 679.7 633.5 605.9 728.3 704.7 742.3 13.41 <0.0001 C* ADF 434.1 425.8 412.3 392.6 477.1 469.5 473.8 11.21 0.0002 C* Lignin (sa) 70.5 79.0 73.2 64.6 96.5 96.4 86.8 5.35 0.0021 L* IVOMD 485.2 488.2 490.4 478.0 472.5 458.4 437.7 5.39 <0.0001 Q* Fermentation process, g/kg of DM Acetic acid 133.7 39.0 31.9 35.4 71.8 90.1 127.3 21.60 0.0034 Q* pH 3.26 3.26 3.33 3.41 3.27 3.29 3.16 0.03 0.0003 Q** Gas losses 361.4 328.2 289.5 259.1 338.9 323.9 329.9 6.59 0.0047 C* DM recovery 637.5 670.6 709.4 740.0 660.2 675.4 669.2 15.88 0.0047 C* Aerobic exposure Initial T, ◦C 28.3 28.2 27.9 28.1 28.0 28.1 28.1 0.18 0.8191 NS Maximum T, ◦C 44.8 42.4 44.1 46.0 42.1 43.1 43.3 0.39 <0.0001 L* Heating rate, ◦C/h 0.60 0.30 0.43 0.56 0.15 0.52 0.28 0.07 0.0009 L* SUM 5, ◦Cc 24.9 26.3 24.1 27.9 21.0 26.0 24.0 1.78 0.1846 NS SUM 9, ◦Cc 38.2 40.5 43.1 42.6 42.4 37.0 32.0 3.52 0.4207 NS Aerobic stability, h 14.7 33.3 34.8 29.5 36.7 21.0 33.0 2.29 <0.0001 C** a DM = dry matter; CP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber; IVOMD = in vitro organic matter digestibility. b NS = no significant; L = linear effect; Q = quadratic effect; C = cubic effect. c Sum of the difference of temperatures between silages and ambient after 5 and 9 d of aerobic exposure. * P < 0.05. ** P < 0.01. 3. Results 3.1. Trial I 3.1.1. Characteristics of sugarcane post-burning All variables were affected by the number of days post-burning, except the coefficients of IVOMD (P > 0.05; Table 1). The DM content had a quadratic response, and the greatest (P < 0.01) value was observed 1-d post-burning. Ash content had a large range during the evaluated times, and the least and greatest values (P < 0.01) were found 10- and 20-d post-burning, respectively. A quadratic response was observed for CP, and the greatest content (P < 0.01) was observed 30-d post-burning. The NDF, ADF, and lignin contents showed a quadratic response; the least values (P < 0.01) were found between 1- and 10-d post-burning. All characteristics associated with sugars showed cubic responses (Table 1); the values of brix, sucrose, pooled (total) sugars, and purity were greatest (P < 0.01) for 10-d post-burning, whereas at this time the redox sugar had its least value. There was a large range (cubic response) in yeast populations, and the least value (P < 0.01) was observed 1-d post-burning (4.47 cfu/g of burned sugarcane), whereas the greatest value (7.11 cfu/g of burned sugarcane) was observed 25-d post- burning. 3.1.2. Chemical composition and fermentation profile of burned sugarcane silages All variables were affected by the time between burning and ensiling (Table 2). DM content exhibited a quadratic response, where the greatest value (P < 0.01) was obtained 25-d post-burning. Ash also showed a large range, and the greatest value (P < 0.01) was found 20-d post-burning. CP showed a quadratic response, and the silage prepared 30-d post-burning exhibited the greatest content (P < 0.01). Considering overall means, burned sugarcane silages showed an increase in CP (43.6 g/kg of DM) compared with the material prior to ensiling (29.2 g/kg of DM). Lesser quantities of NDF, ADF, and lignin were found 15-d post-burning (P < 0.01). Despite the quadratic response, the coefficients of IVOMD were greater (P < 0.01) on 1-, 5-, and 10-d post-burning. In the fermentation profile, acetic acid showed a quadratic response, and the greatest values (P < 0.01) were found in 1- and 30-d post-burning silages (Table 2). Comparison of the means of the 1- and 30-d silages with the means of the other silages revealed that the production of acetic acid was greater in the 1- and 30-d silages (130.5 g/kg of DM) than in other silages (53.6 g/kg of DM). The least pH value was obtained in the 30-d post-burning silage (P < 0.01); however, this value was remarkably low for all silages regardless of the number of post-burning days. Silage prepared 1-d post-burning had the greatest (P < 0.01) gas losses and least (P < 0.01) DM recovery (361.4 and 637.5 g/kg of DM). The least gas losses and greatest DM recovery was observed in 15-d post-burning silage (reduction of 29.5 g/kg of DM on gas losses and increment of 102.5 g/kg of DM on DM recovery compared to 1-d silage). A.P.T.P. Roth et al. / Animal Feed Science and Technology 216 (2016) 68–80 73 25 27 29 31 33 35 37 39 41 43 45 0 20 40 60 80 100 120 140 160 180 200 220 Te m pe ra tu re , º C Aerobic exposu re, h Ambient + 2ºC 1-d 5-d 10-d 15-d 20-d 25-d 30-d Fig. 1. Temperature of burned sugarcane silages prepared in different days post burning during aerobic exposure. 2.5 3.5 4.5 5.5 6.5 7.5 8.5 0 1 2 3 4 5 6 7 8 9 pH Aerobic exposu re, d 1-d 5-d 10-d 15-d 20-d 25-d 30-d F b 3 e t q v w f 3 3 w l l i C r o f 1 ig. 2. pH values of burned sugarcane silages during aerobic exposure (effect of days post burning: P < 0.01; aerobic exposure days: P < 0.01; interaction etween the factors: P < 0.01). .1.3. Aerobic stability of burned sugarcane silages The initial temperature and sum of the differences between silage and ambient temperatures after 5 and 10 d of aerobic xposure were unaffected (P > 0.05) by treatments (Table 2). However, the 20-d post-burning silage had the least maximum emperature and heating rate (P < 0.01), as well as the greatest (P < 0.01) aerobic stability. All silages lost aerobic stability uickly and had temperatures at least 2 ◦C above the ambient temperature after 35 h of aerobic exposure (Fig. 1). An interaction (P < 0.01) between the time before ensiling and the number of aerobic exposure days was observed for pH alues (Fig. 2). Until 6 d after the silos were opened, the 20-d post-burning silage had the least pH values, which is consistent ith the greater aerobic stability observed in this silage. However, after 9 d of aerobic exposure, the least pH value was ound in 10-d post-burning silage. .2. Trial II .2.1. In natura and burned sugarcane treated with different lime levels prior to ensiling Interactions (P < 0.01) between forage type (in natura and burned) and lime levels for DM, ash, CP, NDF, and ADF contents ere observed (Table 3). As expected, in natura sugarcane had greater (P < 0.01) DM content than burned sugarcane. However, ime decreased the DM when applied to in natura sugarcane. For burned sugarcane, silages treated with 15 and 20 g/kg of ime had greater DM content than those treated with 5 and 10 g/kg. Conversely, the ash content quadratically increased with ncreased lime addition, and the greatest value was found in in natura sugarcane treated with 20 g/kg of lime. Overall, CP content quadratically decreased due to the addition of lime (Table 3). For in natura sugarcane, the reduction in P was 4.1 g/kg of DM compared to untreated sugarcane and that treated with 20 g/kg of lime (29.2 g/kg of DM). A greater CP eduction (5.8 g/kg of DM) was observed for burned sugarcane compared to sugarcane untreated and treated with 10 g/kg f lime. Lime quadratically reduced the NDF content in both sugarcanes (Table 3). A reduction of 136.2 g NDF/kg of DM was found or in natura sugarcane when treated with 20 g/kg of lime compared to untreated forage. The burned sugarcane treated with 0 g/kg had 42.2 g NDF/kg of DM less compared to untreated burned sugarcane. Application of 20 g/kg lime quadratically 74 A.P.T.P. Roth et al. / Animal Feed Science and Technology 216 (2016) 68–80 Table 3 Chemical composition (g/kg of DM) of in natura and burned sugarcane either untreated or treated with lime (g/kg of sugarcane, on an as-is fresh matter basis) before ensiling. Item1 In natura Burned SEM P-value2 Contrast3 0 5 10 15 20 0 5 10 15 20 S L S × L Main Interaction DM, g/kg as fed 327.8a 321.0ab 313.9bc 309.9cd 303.4d 260.8ef 256.1f 256.2f 265.7e 266.4e 3.04 <0.0001 0.0417 <0.0001 L** Ash 28.2f 42.9e 64.8d 96.3b 117.6a 17.5g 46.5e 73.9c 93.2b 97.2b 3.17 0.0938 <0.0001 <0.0001 Q** CP 33.3ab 34.5a 34.0ab 31.7b 29.2c 29.2c 26.1d 23.4e 25.3de 26.2d 0.76 <0.0001 0.0007 0.0002 Q** NDF 607.0a 614.2a 573.0b 531.8c 470.8d 407.4e 379.5fg 365.2g 392.7ef 397.2ef 9.27 <0.0001 <0.0001 <0.0001 Q** ADF 329.2ab 335.8a 316.8bc 299.4c 267.8d 245.5e 232.9e 228.2e 240.3e 233.6e 6.25 <0.0001 <0.0001 <0.0001 Q** Lignin (sa) 47.0 54.5 46.1 47.4 35.9 27.2 26.0 25.8 27.6 19.3 2.86 <0.0001 0.0060 0.3685 Q* (a–g) Means in the same row with different superscripts differed (P < 0.05). 1 DM = dry matter; CP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber. 2 S = silage; L = level of lime; S × L = interaction between factors. 3 L = linear effect; Q = quadratic effect. * P < 0.05. ** P < 0.01. reduced ADF content by 61.4 g/kg of DM in in natura sugarcane. However, lime had no effect on ADF content in burned sugarcane where values remained constant. Although there was no interaction (P > 0.05) between silage type and lime levels observed for lignin content, the in natura sugarcane exhibited greater (P < 0.01) lignin content than burned sugarcane (Table 3). Overall, the greatest lime level (20 g/kg) reduced the lignin content by 11.1 and 7.9 g/kg of DM when applied on in natura and burned sugarcane compared to the respective untreated forages (P < 0.05). 3.2.2. Chemical composition and fermentation profile of in natura and burned sugarcane silages treated with different lime levels All variables were affected by the interaction (P < 0.05) between silage type and lime levels, as shown in Table 4. In natura silages had greater (P < 0.01) DM content than burned silages. However, in natura silages showed no change in DM content due to the lime application, whereas there was a quadratic increase in DM content in burned silages following lime application. Ash content exhibited a similar response; in other words, a quadratic increase (P < 0.01) in ash content following lime application was observed in both types of silage; in general, in natura silages had greater ash contents than burned silages. A quadratic reduction (P < 0.01) in CP content was observed following lime application, regardless of silage type (Table 4). The greatest values of CP were observed in untreated in natura sugarcane or that treated with 10 g/kg of lime and also in untreated burned silage. Overall, in natura silages exhibited greater (P < 0.01) NDF contents than burned silages (Table 4). There was a quadratic reduction in NDF content with increased lime addition for both in natura and burned silages. If untreated silages are used as a baseline, lime reduced the NDF content by 143.2 and 265.1 g/kg of DM when in natura silage was treated with 20 g/kg lime and burned silage was treated with 10 g/kg, respectively. Likewise, these also were the most effective treatments for reducing (P < 0.01) the ADF content. Addition of lime led to a marginal and quadratic decrease in lignin content (P < 0.05) of in natura silage, and the application of 10 and 20 g/kg of lime led to also a quadratic decrease in lignin content of burned silages. All burned silages (except those treated with 5 g/kg of lime) produced more (P < 0.01) acetic acid than in natura silages revealing overall means of 47.0 vs. 25.6 g/kg of DM, respectively (Table 4). Lime was more effective in increasing the production of acetic acid in in natura silages, but only when applied at great concentrations (15 and 20 g/kg). There was a difference (P < 0.01) in pH values between silage types only when great concentrations of lime (15 and 20 g/kg) were used in the treatment of silages; in natura silages had greater pH than burned silage (Table 4). However, as expected, pH cubically increased with an increase in the concentration of lime applied. The gas losses of in natura silages quadratically increased (P < 0.01) with lime addition; comparing untreated silage with those treated with 15 and 20 g/kg of the additive, the increase was 44.8 g/kg of DM (Table 4). Conversely, the use of lime quadratically reduced the gas losses in burned silages, especially when used at 10 g/kg (103.5 g/kg of DM less than untreated silage). Overall, lime addition quadratically decreased (P < 0.01) DM recovery in in natura silages (except the level of 5 g/kg, which increased this variable), whereas the opposite effect was observed for burned silages. 3.2.3. Aerobic stability of in natura and burned sugarcane silages treated with different lime levels All variables related to aerobic stability were changed (P < 0.05) by the interaction between silage type and lime levels, as shown in Table 4. Overall, lime had the desired effect on initial temperature, decreasing (P < 0.05) this variable for both silage types, especially when great levels were used (15 and 20 g/kg). Similarly, the addition of lime reduced (P < 0.05) the maximum temperature, particularly at 15 g/kg for both silage types. Lesser (P < 0.05) heating rates were observed when in natura and burned silages were treated with 15 and 20 g/kg of lime, as well as for in natura silage treated with 10 g/kg. Similar results were found for SUM 5 and SUM 9 (P < 0.01). Consequently, aerobic stability increased (P < 0.01) when silages A .P.T.P. R oth et al. / A nim al Feed Science and Technology 216 (2016) 68–80 75 Table 4 Chemical composition, fermentation, and aerobic stability of in natura and burned sugarcane silages untreated or treated with lime (g/kg of sugarcane, on an as-is fresh matter basis). Item1 In natura Burned SEM P-value2 Contrast3 0 5 10 15 20 0 5 10 15 20 S L S × L Main Interaction Chemical composition, g/kg of DM DM, g/kg as fed 272.7a 269.8a 259.0a 254.4a 265.1a 170.8d 202.6c 214.9bc 226.3b 212.2bc 7.43 <0.0001 0.2014 0.0007 Q* Ash 34.2f 48.5e 67.7d 108.8b 140.8a 23.4g 46.3e 65.7d 96.1c 112.5b 2.70 <0.0001 <0.0001 0.0007 Q** CP 42.5a 38.6b 43.6a 38.1b 37.3b 43.2a 34.5c 29.7e 30.8de 32.5d 0.95 <0.0001 <0.0001 <0.0001 Q** NDF 755.5a 689.0bc 708.7b 683.6c 612.3d 681.1c 506.7e 416.0g 440.9f 443.8f 10.04 <0.0001 <0.0001 <0.0001 Q** ADF 426.2a 386.7b 402.4b 416.3a 392.9b 428.3a 310.1c 277.0d 302.5c 310.0c 6.03 <0.0001 <0.0001 <0.0001 Q** Lignin (sa) 53.0ab 46.7b 48.7b 56.4ab 44.6b 67.0a 35.5bc 24.6c 39.6bc 29.2c 5.71 0.0077 0.0029 0.0393 Q* Fermentation process, g/kg of DM Acetic acid 21.6c 18.0c 13.4d 36.0b 38.9b 50.7a 39.9b 50.4a 48.7a 45.2ab 2.63 <0.0001 <0.0001 <0.0001 Q* pH 3.42f 3.66e 4.18d 5.52b 5.98a 3.31f 3.65e 4.11d 4.48c 4.58c 0.05 <0.0001 <0.0001 <0.0001 C** Gas losses 149.2cd 83.2f 156.6c 227.1b 238.4b 272.2a 122.1de 111.9e 149.4de 145.8de 10.80 0.1028 <0.0001 <0.0001 Q** DM recovery 849.5cd 912.0a 842.1d 771.6e 760.3e 727.0f 877.4bc 887.8b 850.1bc 853.6bc 10.96 0.1028 <0.0001 <0.0001 Q** Aerobic exposure Initial T, ◦C 25.3a 24.5bc 25.5a 24.8b 24.8b 24.8b 24.5bc 24.3c 24.2c 24.3c 0.14 <0.0001 0.0016 0.0220 C*** Maximum T, ◦C 42.2a 40.8a 30.8bc 29.5c 30.2c 42.2a 41.0a 37.7b 29.8c 34.5b 1.05 0.0033 <0.0001 0.0196 C* Heating rate, ◦C/h 0.18bc 0.16c 0.05d 0.05d 0.06d 0.22ab 0.27a 0.13c 0.04d 0.07d 0.02 0.0008 <0.0001 0.0252 C* SUM 5, ◦C4 15.0c 11.4d −0.8e −5.1ef −0.2e 29.5a 24.1b −0.2e −7.8f −6.6f 1.67 0.0033 <0.0001 <0.0001 L** SUM 9, ◦C4 21.9bc 20.8bc 10.3c −8.4d 0.1c 58.7a 38.7b 20.8bc −10.0d 4.4c 4.01 0.0003 <0.0001 <0.0001 L** Aerobic stability, h 75.0c 83.0c 194.5a 214.0a 214.0a 36.3d 49.0d 95.0c 204.3a 153.0b 8.12 <0.0001 <0.0001 0.0009 C*** (a–g) Means in the same row with different superscripts differed (P < 0.05). 1 DM = dry matter; CP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber. 2 S = silage; L = level of lime; S × L = interaction between factors. 3 L = linear effect; Q = quadratic effect; C = cubic effect. 4 Sum of the difference of temperatures between silages and ambient after 5 and 9 d of aerobic exposure. * P < 0.05. ** P < 0.01. *** P < 0.10. 76 A.P.T.P. Roth et al. / Animal Feed Science and Technology 216 (2016) 68–80 23 25 27 29 31 33 35 37 39 41 43 0 20 40 60 80 10 0 12 0 14 0 16 0 18 0 20 0 220 Te m pe ra tu re , º C Ambient + 2ºC 0 g/kg 5 g/kg 10 g/kg 15 g/kg 20 g/kg 23 25 27 29 31 33 35 37 39 41 43 0 20 40 60 80 10 0 12 0 14 0 16 0 18 0 20 0 220 Te m pe ra tu re , º C Aerobic exposure, h Ambient + 2ºC 0 g/kg 5 g/kg 10 g/kg 15 g/kg 20 g/kg a b Fig. 3. Temperature of in natura (a) and burned (b) silages either untreated or treated with lime (g/kg of sugarcane, on an as-is fresh matter basis) during aerobic exposure. were treated with great levels of lime. In natura silages treated with 10, 15, and 20 g/kg lime and burned silage treated with 15 g/kg lime exhibited great aerobic stability (>8 d). Conversely, untreated silages or those treated with less levels of lime exhibited large peaks of temperature during aerobic exposure (Fig. 3). During aerobic exposure, pH was affected by interactions (P < 0.01) among silage type, lime levels, and time exposed to oxygen (Fig. 4). For the first 3 days of aerobic exposure, both silages exhibited greater pH values when treated with 15 and 20 g/kg of lime. However, after day 3, the most effective treatments were the application of 10 and 15 g/kg of lime for in natura and burned silages, respectively. 4. Discussion 4.1. Trial I Prior to ensiling, the burned sugarcane had a reduction in sucrose content with increases in redox sugars over time while remained in the field likely by invertase activity. Invertase activity can convert sucrose into glucose and fructose during the accumulation of sucrose in tissues of sugarcane (Whittaker and Botha, 1997), and its activity may increase in great temperatures (e.g., sugarcane burning). This hypothesis could explain why sugarcane purity also decreased, taking into consideration the quantity of sucrose in relation to total solids. Even with cubic effect, we observed that delay in post-burning ensiling increased yeast population in sugarcane likely by exudation or escape of sugars or both, caused by burning, which destroy wax layer that enfolds the cell-wall and cause cracks in the stalk (Bernardes et al., 2007). A previous study also reported increases of yeast population in burned sugar- cane throughout the 14-d post-burning (Roth et al., 2010). In our study, increases in yeast population may be the cause of the increased fiber fraction in sugarcane that remained in the field for many days post-burning. Yeasts use water-soluble carbohydrates (WSC) as the main substrate for their growth; in turn, the fiber fraction increases via the concentration effect (Rabelo et al., 2014). However, the increases in NDF content unaffected IVOMD of burned sugarcane for unknown reasons. DM content also increased according to the length of time the sugarcane remained in the field post-burning likely because the increased yeasts activity, and water loss caused by burning. CP decreased up to 10-d post-burning and increased after this time. Yeasts and other microorganisms are composed of protein, and certainly their overgrowth after 10-d post-burning contributed to increase the CP content of sugarcane. When the silos were opened, great amounts of acetic acid was observed in the 1- and 30-d post-burning silages likely arising the better conditions found by LAB population to growth. Acetic acid is a powerful antifungal agent (Danner et al., 2003) capable of inhibiting yeast and reducing ethanol formation and DM loss during silage fermentation (Ávila et al., 2009). A.P.T.P. Roth et al. / Animal Feed Science and Technology 216 (2016) 68–80 77 2.5 3.5 4.5 5.5 6.5 7.5 8.5 0 1 2 3 4 5 6 7 8 9 pH 0 g/kg 5 g/kg 10 g/kg 15 g/kg 20 g/kg 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 0 1 2 3 4 5 6 7 8 9 pH Aerobic exposu re, d 0 g/kg 5 g/kg 10 g/kg 15 g/kg 20 g/kg a b F a T a t t A i a l s s t i t w q d a e l p c m S ( ig. 4. pH values of in natura (a) and burned (b) silages either untreated or treated with lime (g/kg of sugarcane, on an as-is fresh matter basis) during erobic exposure (effect of silage type: P > 0.10; lime: P < 0.01; aerobic exposure days: P < 0.01; interaction among the factors: P < 0.01). here was not a clear effect of post-burning days on DM recovery (cubic effect); however, despite of the greater acetic cid concentration, 1- and 30-d post-burning silages did not have greater DM recovery. The 15-d post-burning silage had he greatest DM recovery, possibly due to decreased yeast activity because this silage had less sugar contents compared to hose that remained in the field for fewer days post-burning. Conversely, all silages had low DM recovery (<750 g/kg of DM). lthough all silages exhibited low pH (<3.45), this does not ensure a desirable fermentation, as yeasts are able to growth n a wide range of pH, from 2.5 to 8.5 (Orij et al., 2011). The main metabolic pathway of yeast uses pyruvate decarboxylase cetaldehyde and the posterior reduction of acetaldehyde into ethanol occurs (Rooke and Hatfield, 2003), resulting in DM osses of 489 g/kg as fed during fermentation (McDonald et al., 1991). Indeed, a low DM recovery has been reported in burned ilages because the intense yeasts activity (Roth et al., 2010; Siqueira et al., 2010). The great DM losses also explain the reduction in DM content of silages compared to sugarcane before ensiling in our tudy, a response previously reported by Siqueira et al. (2007). Similarly, ash, CP, NDF, and ADF increased in silages compared o forage. Fermentation process occurs by converting of soluble sugars to organic acids, and an increase in other compounds s expected due to the concentration effect (Rabelo et al., 2014). Furthermore, lignin content of burned silages linearly increased with increased post-burning days likely as a response for he yeasts overgrowth. The 1-, 5-, and 10-d burned silages had lesser lignin content when compared with those produced ith sugarcanes that remained a long time in the field post-burning. Thus, the lesser lignin contents probably explain the uadratic response observed for the coefficients of IVOMD, where burned silages up to 10-d post-burning had a greater igestibility. The negative effect of lignin on digestibility has been previously documented (Jung and Allen, 1995). However, ll silages had low digestibility (435–490 g/kg of organic matter), and our data are in accordance with previous studies (Roth t al., 2010; Siqueira et al., 2011b), which also reported a low digestibility of burned sugarcane silages. The silage produced from sugarcane that remained in the field 15-d post-burning had the greatest maximum temperature ikely by the greater DM recovery. Silages with great DM recovery often exhibit a desirable fermentation pattern (i.e., greater roduction of lactic acid and preservation of residual WSC) and can have lesser aerobic stability (McDonald et al., 1991), as onfirmed in this study. Aerobic exposure of silage leads to extensive oxidization of WSC driven by the growth of epiphytic icroorganisms, especially yeasts, with increases in temperature and decreases in the nutritive value of feed (Kung and tanley, 1982; Wilkinson and Davies, 2012). The 1-d post-burning silage exhibited the greatest production of acetic acid; however, this silage had low aerobic stability <15 h). Although acetic acid has been strongly related to enhance aerobic stability (Danner et al., 2003), yeasts are able to 78 A.P.T.P. Roth et al. / Animal Feed Science and Technology 216 (2016) 68–80 growth in a wide range of conditions (Orij et al., 2011; Pahlow et al., 2003). Conversely, the 20-d post-burning silage showed the greatest aerobic stability, as well as the least pH after 6 d of aerobic exposure. This is probably due to lesser sugar content during ensiling compared to those silages prepared with sugarcane that remained in the field for a shorter time post-burning. The lesser pH found in the 20-d post-burning silage during aerobic exposure is likely associated with the lesser yeasts growth. Yeasts are able to use lactic acid as a substrate, causing an increase in pH values, especially when the pH is greater than 4.5, favoring the growth of other spoilage microorganisms (Wilkinson and Davies, 2012). Consequently, there is an important reduction in the nutritive value of silage. Overall, although ensiling of burned sugarcane between 10- to 15-d post-burning revealed a greater DM recovery, in our study there was not a clear effect of post-burning days on the fermentation patterns and aerobic stability of silages, since a cubic response occurred for many variables. Likewise, Roth et al. (2010) found inconsistent results in silages produced with burned sugarcane throughout the 14-d post burning. Brazil is the largest producer of sugarcane in the world (FAO, 2015) and accidental fire still occurs in the field, but the quality of burned sugarcane silage has been few investigated, and further studies are necessary to understand the impact of burning on it. 4.2. Trial II In natura sugarcane had greater DM, CP, and fiber content than burned sugarcane, both before and after ensiling. During burning, straw is the primary part of the plant affected; straw has great DM and fiber content and a less amount of CP. The loss of straw, which comprises mostly senescent leaves that contain great NDF content, during burning, explains our results. A previous study also reported similar results in in natura and burned sugarcane (Siqueira et al., 2010). Ash content quadratically increased in in natura and burned sugarcane before and after ensiling by lime addition, once this additive has great ash content; however, this response was not translated in marked increases in DM content likely by water loss caused by heating of sugarcane when lime was added. Calcium oxide in contact with water produces an alkaline compound (CaO + H2O → Ca(OH)2 + heat), and considerable quantity of heat (15,300 cal/mol) may be produced. In addition, CP quadratically reduced with increasing additions of lime, which is probably due to a dilution effect. Overall, the fiber fraction quadratically decreased by lime addition, and the greatest levels (i.e., 15 and 20 g/kg) reflected in lesser values. Alkaline additives have been used in order to reduce the fiber fraction and increase the fiber digestibility, or both (Santos et al., 2009). Lime often has the capacity to expand the fiber (hydrolysis), causing a rupture in the ester linkages between lignin and hemicellulose, and breaking the hydrogen linkages between cellulose and hemicellulose (Klopfenstein, 1980). Thus, our results are in accordance with the chemical role played by lime. Despite of cubic effect, lime addition increased the pH values for both silages; however, pH increased more strongly in in natura silages than burned silages. Increases in pH values are expected because the application of lime to sugarcane causes hydration of calcium oxide and production of alkali (Daniel et al., 2013). Acetic acid concentration increased in in natura silages due to the application of lime. If lime is able to break linkages between lignin and hemicellulose (Klopfenstein, 1980), an increase in the production of organic acids is expected (verified for acetic acid in the present study) because LAB metabolize nonstructural carbohydrates into organic acids (Rooke and Hatfield, 2003). Moreover, LAB (mainly heterofermentative LAB) probably had greater activity in elevated-pH conditions, since these microorganisms growth slowly when pH values are below 3.5 (McDonald et al., 1991). A quadratic response was observed for gas losses, and great levels of lime (15 and 20 g/kg) led to an increase in gas losses in in natura silages, resulting in lesser DM recovery (except for silage treated at 5 g/kg). This may be explained by an increase in acetic acid production because there is production of CO2 in heterolactic pathway caused by the fermentation of WSC, and this via is less efficient than the homolactic pathway in DM recovery (Muck, 2010). Additionally, the reduction in DM recovery in in natura silages with increased lime application likely suggest greater ethanol production (not measured in our study), which is the major problem in sugarcane silages (Kung and Stanley, 1982). Greater DM recovery is not always observed when using great doses of alkaline additives (Pedroso et al., 2007), which is consistent with our study. An opposite response in DM recovery due to the lime addition was observed in burned silages. Lime quadratically increased DM recovery in burned silages due to the lesser gas losses. Yeasts use WSC producing ethanol and CO2 (McDonald et al., 1991). A great and positive correlation (R2 = 0.89) between ethanol and gas losses has been observed (Pedroso et al., 2005). In this case, the great production of CO2 is likely due to the low efficient metabolic pathway of yeasts, once acetic acid level is no longer increased by the application of lime. Our results are consistent with the greater DM recovery observed in other cultivars of sugarcane ensiled with lime (Santos et al., 2008). Application of lime (>10 g/kg) reduced the heating rate in silages, thereby these silages exhibited a long period of stability when aerobically exposed. Lime possesses antifungal properties, and the inhibition of spoilage microorganisms must be related to changes in the osmotic potential of silage that reduce the growth of yeast (Pahlow et al., 2003). The positive effect of lime to enhance aerobic stability has been reported in several studies (Amaral et al., 2009; Balieiro Neto et al., 2009; Rezende et al., 2011). Succession of yeast species occurs from the anaerobic phase to the aerobic phase when silos are opened (Pahlow et al., 2003), and this process continues during the aerobic exposure phase (Pitt et al., 1991). A majority of yeast species that are active during oxygen exposure are the same epiphytic species found in the forage before ensiling (Pahlow et al., 2003). Increased aerobic stability should be expected in burned sugarcane because heat could interfere with the succession process (Siqueira et al., 2010). However, taking into consideration the overall mean, the in natura silages had great stability compared t o e b h t e 5 c t m f f A A h R Á A A B B B B C D D F J K K K K M M M M M M M M A.P.T.P. Roth et al. / Animal Feed Science and Technology 216 (2016) 68–80 79 o burned silages (156.1 vs. 107.5 h, respectively). Our hypothesis is that burned sugarcane probably had greater production f lactic acid (which was not measured in this study) suggested by the great DM recovery. Lactic acid no has an antifungal ffect per se (Moon, 1983), and can be largely used as a substrate for the growth of yeast (Wilkinson and Davies, 2012). Thus, etter fermentation usually results in low aerobic stability of silages (McDonald et al., 1991). Linear decreases in accumulated temperature of in natura and burned silages were observed; however, lime addition ad a cubic effect on aerobic stability. Different lime levels led to inconsistent results, mainly when considered the different ypes of sugarcane (in natura and burned). Conversely, the greater levels of lime (15 and 20 g/kg) seems to be consistent to nhance aerobic stability of silages, regardless of sugarcane type. . Conclusions The impact of post-burning days on the characteristics of burned silage was not clear, and further studies should be onsidered. However, considering the approach in which this study was carried out, if a sugarcane field is burned accidentally, he farmers have 10–15 d time period to ensile the forage so that the digestibility and conservation of nutrients may be aximized. Lime consistently improved DM recovery in burned silages. The greater levels of lime (15 and 20 g/kg) reduced fiber raction and enhanced aerobic stability for both in natura and burned silages. In summary, lime may be used as an additive or both in natura and burned silages since in greater levels. cknowledgement The authors wish to express their appreciation to the Sao Paulo Research Foundation (FAPESP) for their financial support. ppendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at ttp://dx.doi.org/10.1016/j.anifeedsci.2016.03.010. eferences vila, C.L.S., Pinto, J.C., Figueiredo, H.C.P., Schwan, R.F., 2009. 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