Fermentative characteristics and aerobic stability of sorghum silages containing different tannin levels

Animal Feed Science and Technology 154 (2009) 1–8

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Fermentative characteristics and aerobic stability of sorghum silages containing different tannin levels Simone Gisele de Oliveira a,∗, Telma Teresinha Berchielli b, Ricardo Andrade Reis b, Maria Eliane Vechetini b, Márcio dos Santos Pedreira c a b c

Universidade Federal do Paraná, Rua dos Funcionários, 1540, CEP 80035-050, Curitiba, PR, Brazil Faculdade de Ciências Agrárias e Veterinárias - Unesp, Jaboticabal, SP, Brazil Universidade do Sudoete da Bahia, Itapetinga, BA, Brazil

a r t i c l e

i n f o

Article history: Received 9 September 2008 Received in revised form 30 June 2009 Accepted 3 July 2009

Keywords: Conserved forage Fermentation Polyethylene glycol Polyphenols

a b s t r a c t Two experiments were conducted to evaluate effects of tannin levels in sorghum silages supplemented, or not, with polyethylene glycol (PEG) on silage chemical composition and fermentative characteristics as well as aerobic stability of silages supplemented with urea or concentrate. A completely randomized experimental design was applied in both experiments. Silages without PEG had higher CP levels and lower ammonia N concentration (81.8 g/kg DM and 55.3 g/kg N, respectively) as compared to silages with PEG (70.6 and 63.4, respectively). High-tannin sorghum levels were not able to maintain silage aerobic stability, probably due to their low concentrations in the silages. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Tannins are defined as high molecular weight phenolic compounds with the ability to bind to macromolecules such as proteins, structural carbohydrates and starch, thereby impairing their degradation (McSweeney et al., 2001; Silanikove et al., 2001). Tannins are divided into two main groups, being hydrolysable and condensed. The polyphenols found in sorghum are condensed tannins (Jansman, 1993).

Abbreviations: ADFom, acid detergent fiber; ADIN/N, acid detergent insoluble N; aNDFom, neutral detergent fiber; CP, crude protein; DM, dry matter; HT, high tannin; LT, low tannin; PEG, polyethylene glycol. ∗ Corresponding author. Tel.: +55 41 3353 6492; fax: +55 41 3252 4149. E-mail addresses: [email protected], [email protected] (S.G. de Oliveira). 0377-8401/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2009.07.003

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Tannin content and chemical structure determine if their effects on animal metabolism are beneficial or harmful (Barry and McNabb, 1999; Schofield et al., 2001). However, during silage fermentation, the presence of tannins is considered an advantage because they protect forage proteins from degradation (Salawu et al., 1999; Kondo et al., 2004) by inhibiting plant and microbial enzymes and/or by forming complexes with proteins (Makkar, 2003). These silages containing tannins lose less N as ammonia is well documented (e.g., Gonc¸alves et al., 1999; Adesogan and Salawu, 2002), showing that they effectively reduce protein degradation. Sorghum hybrids have different tannin levels, which may play an important role in sorghum resistance to bird attacks (Beta et al., 2000) and to the presence of molds before and after grain maturation (Mullins and NeSmith, 1988). Exposure to oxygen causes temperature increases and aerobic deterioration of silages, and decreases their nutritional value due to growth of undesirable microorganisms and synthesis of toxins (Danner et al., 2003; Filya, 2004). Few studies have evaluated the aerobic stability of sorghum silage vis-à-vis the presence of tannins, evidencing the need for further studies. Polyethylene glycol (PEG) has been commonly used in studies to determine effects of tannins on silage preservation and animal metabolism (Villalba and Provenza, 2002; Ben Salem et al., 2003), as PEG binds to tannins to inhibit their biological action. The objective was to evaluate effects of tannins on chemical composition and fermentation characteristics of sorghum silages with or without PEG addition. A second experiment was completed to determine aerobic stability of diets containing sorghum silage, with different tannin contents, with supplementation (or not) of urea, or a mixture of corn grain and soybean meal aiming at simulating effects of aerobic degradation of complete rations. 2. Material and methods Field studies were conducted at the Experimental Farm of Faculdade de Ciências Agrárias e Veterinárias – UNESP, Brazil, 21◦ 15S, 48◦ 19 E, 605 m altitude, from November 2002 to June 2003. Two hybrid sorghum varieties were used (Table 1), being silage sorghum 1F305 from Dow Agrosciences (Indianapolis, IN, USA), and forage sorghum BR 700 produced by EMPRAPA (Sete Lagoas, MG, Brazil). Two areas measuring two hectares were cultivated. The areas were fertilized at seeding with 350 kg/ha of a formulation 10:20:20 of N, phosphorus (P2 O5 ) and potassium (K2 O). There was no topdress application of fertilizer. Sorghum hybrids were harvested at 14 cm, when forage mass contained 330–350 g/kg dry matter (DM), with an ensiling machine regulated to a 1–1.5 cm particle size. The harvested material was stored and compacted with the aid of a tractor in trench silos in Experiment 1, and in PVC silos in Experiment 2. 2.1. Experiment 1 PEG effects on fermentation were evaluated in both sorghum hybrids. The treatments were: lowtannin sorghum silage without PEG (LTS), low-tannin sorghum silage with PEG (LTSP), high-tannin sorghum silage without PEG (HTS), and high-tannin sorghum silage with PEG (HTSP). The PEG solution was prepared according to Ben Salem et al. (1999) in which 90 g of PEG (MW: 4000 Da) were diluted in 300 mL of water per kg of ensiled DM. The solution was sprayed onto the cut material before ensiling. Treatments without PEG addition were sprayed with 300 mL of water to simulate effects of the higher moisture of the material sprayed with PEG on the evaluated parameters. A total number of 16 silos, four silos per treatment, were made of polyvinyl chloride (PVC), and measured 10 cm in diameter and 50 cm in length. Compaction was to a density of 700 kg/m3 , and the silos were closed using PVC lids with Bunsen valves. Silos were opened 120 days after ensiling, and material was homogenised and one sample was pressed using a hydraulic press (MA-098, Marconi, Piracicaba, SP, Brazil) to extract silage juice. The pH was immediately measured using a pH meter (Accumet, model HP-71, Fisher Scientific, Pittsburgh, PA, USA), and the silage juice was stored in a freezer at −20 ◦ C. After thawing, samples were centrifuged at 15,000 × g for 15 min at 4 ◦ C, and the supernatant fraction was analysed for NH3 –N concentrations as described by Chaney and Marbach (1962), and for volatile fatty acids (VFA) according to Palmquist and Conrad (1971).

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A second aliquot (300 g) was dried in a forced ventilation oven at 55 ◦ C for 72 h, and ground in a Wiley mill (Arthur H. Thomas Co., Philadelphia, PA, USA) through a 1 mm screen in order to determine DM by drying at 105 ◦ C for 12 h in a forced ventilation oven, N content according to method 976.05 of AOAC (1990), and neutral detergent fiber (aNDFom), acid detergent fiber (ADFom), and lignin(sa) according to methodologies adapted from Van Soest et al. (1991). Heat stable amylase and sodium sulphite were used in the aNDFom procedure, and aNDFom and ADFom results are expressed on an ash-free basis. The N concentration in the residues of ADFom analysis was also determined to estimate ADF bound N. Material of both hybrids was freeze-dried and ground using a Wiley mill to 1 mm particles to determine the concentration and the activity of condensed tannins at the Laboratório de Nutric¸ão Animal of the Centro de Energia Nuclear na Agricultura (CENA), USP, in Piracicaba, SP, Brasil. Condensed tannin analysis used whole-plant samples (i.e., panicle, leaves and stem – Experiments 1) and silage samples (Experiment 2) were evaluated by butanol–HCl method according to Porter et al. (1986). A completely randomized design within a 2 × 2 factorial arrangement (i.e., 2 tannin concentration × PEG) was applied. Data were analyzed using PROC GLM of SAS (2001) to evaluate main effects (i.e., tannin levels and additive), and significant interactions were followed-up to determine simple effects, at a 5% significance level using Tukey’s test. 2.2. Experiment 2 Aerobic stability was determined in silages of both sorghum hybrids produced in trench silos. Silos were opened 60 days after ensiling, and silage aerobic stability was evaluated according to methodology described by O’Kiely et al. (1999) as modified by O’Kiely et al. (2002). In order to determine oxygen effects on silages, urea or a concentrate (i.e., corn grain and soybean meal) was added to simulate typical diets fed to beef cattle. Treatments consisted of low-tannin sorghum silage (988.5 g/kg DM) and urea (11.5 g/kg DM) (LTU); low-tannin silage (400 g/kg DM) + concentrate (600 g/kg DM) (LTC); high-tannin sorghum silage 988.5 g/kg DM) and urea (11.5 g/kg DM) (HTU); and high-tannin silage (400 g/kg DM) + concentrate (600 g/kg DM) (HTC). The concentrate consisted of a mixture of corn grain (660 g/kg DM) and soybean meal (340 g/kg DM). On the day that the silos were opened, the above described treatments and 1.2 kg (DM) of both sorghum hybrids supplemented with urea or concentrate (according to treatment) were placed into Styrofoam boxes (40 cm × 25 cm × 25 cm). The temperature of each mixture of silage and concentrate was measured. Ambient temperature was determined by measuring the temperature of water in another box. Temperature readings and box weights were recorded twice daily for 10 days. Daily box weight and DM content (determined according to the methodology described in Experiment 1) were used to calculate DM losses during the evaluation period. Aerobic stability was defined as the time required to increase silage temperature 2 ◦ C above ambient. The parameters used to evaluate aerobic stability during the 10 day period were maximum temperature inside the mass (MT), the number of days required to reach MT, the number of days required to reach mass temperature 2 ◦ C above ambient, and DM losses during the period. Changes in silage chemical composition were determined in the material collected at the beginning (day 0) and end (day 10) of the evaluation period, using DM, CP, aNDFom, ADFom, and N bound to ADFom as described in Experiment 1. A completely randomized design within a 2 × 2 factorial arrangement (i.e., 2 tannin concentration × urea or concentrate) was applied. Data were analyzed using PROC GLM of SAS (2001) to evaluate main effects and significant interactions were followed-up to determine simple effects, at a 5% significance level using Tukey’s test. 3. Results 3.1. Experiment 1 Silages produced with the high-tannin hybrid, and those with no PEG addition, had higher (P<0.05) CP contents compared to the other treatments (Table 2). The aNDFom concentration was impacted by tannin level, where LT sorghum had higher (P<0.05) values compared with HT sorghum.

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Table 1 Chemical composition of the hybrid sorghum plants used in the study. 1F305 (low tannin)

DM (g/kg) CP (g/kg DM) aNDFom (g/kg DM) ADFom (g/kg DM) Condensed tannins (g/kg DM)

BR 700 (high tannin)

Plantsa

Silageb

Plantsa

Silageb

329.3 58.8 600.2 375.2 0.2

380.2 84.3 570.9 283.8 0.2

345.5 63.4 566.1 363.9 5.9

363.5 85.3 547.4 272.4 1.0

MS = dry matter; CP = crude protein; aNDFom = neutral detergent fiber; ADFom = acid detergent fiber. a b

n = 2. n = 4.

Table 2 Chemical composition of silages of sorghum hybrids (low and high-tannin) with or without polyethylene glycol (PEG). LT sorghum

DM (g/kg) CP (g/kg DM) aNDFom (g/kg DM) ADFom (g/kg DM) Lignin(sa) (g/kg DM) ADIN/N (g/kg N)

HT sorghum

SEM

−PEG

+PEG

−PEG

+PEG

309.0b 80.5 590.8 433.9 62.5 184.8

355.3a 64.0 578.9 415.7 65.5 209.5

362.8 83.1 538.7 407.2a 81.7a 238.7 a

332.9 77.2 488.3 315.3b 65.5b 142.3 b

1.88 0.63 3.59 2.13 0.43 1.66

P Sorghum

PEG

S×P

0.122 0.027 0.002 0.001 0.001 0.437

0.402 0.004 0.109 0.082 0.091 0.001

0.002 0.118 0.305 0.005 0.007 0.001

LT = low-tannin sorghum silage; HT = high-tannin sorghum silage. MS = dry matter; CP = crude protein; aNDFom = neutral detergent fiber; ADFom = acid detergent fiber; ADIN/N = acid detergent insoluble N (g/kg N). a,b Means followed by different letters in the same row within response parameter differ (P<0.05).

The DM, ADFom, lignin(sa), and ADIN/N were influenced by the interactions of sorghum silage chemical composition with tannin levels and PEG addition (Table 2), where DM was higher with PEG, but only within LT sorghum, and ADFom, lignin(sa) and ADIN/N were higher with PEG, but only within HT sorghum. NH3 –N concentration in silages was higher (Table 3) when silage fermentation occurred in the presence of PEG (P<0.05) and tended (P=0.056) to be higher in HT sorghum. There were no effects on concentrations of acetate and butyrate, and no propionate was detected in the ensiled material. 3.2. Experiment 2 Forage source affected (P<0.05) both the time required to reach maximum temperature (MT) and DM loss due to air exposure (Table 4), and all silages were very unstable after 3 or 4 days of aerobic exposure. In high-tannin silages (HT), MT was 8 days after the silo was opened whereas in low-tannin silages (LT) MT was at 6 days. Nevertheless, DM loss was higher in HT silages. Concentrate suppleTable 3 Fermentation characteristics of silages of sorghum hybrids (low- and high-tannin) with or without polyethylene glycol (PEG). LT sorghum −PEG pH NH3 –N (g/kg N) Acetate (g/kg DM) Propionate (g/kg DM) Butyrate (g/kg DM)

3.88 52.8 6.1 0 0.3

HT sorghum +PEG

3.94 61.0 6.6 0 0.1

−PEG 3.86 57.7 7.9 0 0.3

SEM +PEG

3.88 65.8 4.7 0 0.1

LT = low-tannin sorghum silage; HT = high-tannin sorghum silage.

0.011 0.160 7.976 – 0.254

P Sorghum

PEG

S×P

0.004 0.056 0.188 – 0.981

0.005 0.004 0.223 – 0.270

0.127 0.983 0.816 – 0.408

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Table 4 Aerobic stability of silages of sorghum hybrids (low- and high-tannin) added urea or concentrate. LT sorghum

HT sorghum

Urea

Concentrate

Urea

Concentrate

6 3 59.3 113

8 4 50.7 210

7 3 57.7 121

Time intervals (days) DMT 4 TE 3 MT (◦ C) 46.0 DML (g/kg DM) 88

SEM

0.6 0.3 1.7 1.8

P Sorghum

Conc.

S×C

0.009 0.170 0.256 0.042

0.419 0.392 0.001 0.268

0.066 0.392 0.062 0.067

LT = low-tannin sorghum silage; HT = high-tannin sorghum silage. DMT = days until maximum temperature; TE = time for temperature increase (2 ◦ C higher than environmental temperature); MT = maximum temperature; DML = dry matter losses.

mentation to the silages increased MT versus urea supplementation. The time required to increase the mass temperature 2 ◦ C above the ambient temperature was not affected by forage tannin level or by supplementation. The effect of aerobic deterioration on dietary chemical composition is in Table 5. CP levels of the LT silage with urea decreased from day 0 to day 10, and were different from the other treatments, where CP level increased from day 0 to day 10. ADFom levels of treatment for low-tannin silage were different among evaluation periods. The ADIN/N level markedly increased, and was significantly different between days 0 and 10 (P≤0.05). 4. Discussion 4.1. Experiment 1 Condensed tannin levels in the forages were low, even in the BR700 hybrid, which is considered a high-tannin forage. Cummins (1971) evaluated sorghum hybrids with different tannin levels and reported that, although the grains have a higher level of tannins, their levels in other parts of the plant should also be considered, which was confirmed by Jambunathan et al. (1986). The low tannin levels in the present study indicate that the presence of tannins in parts of the plant other than grains was probably irrelevant, and that the tannins present in the grains may have been diluted as the analyses were on whole plants. The levels of condensed tannins apparently decreased in the high-tannin hybrid after the fermentation process. Cummins (1971) reported that tannin levels in sorghum grains were reduced between 4 and 10% after silage fermentation, and Osman (2004) observed reductions of 15 to 35% in sorghum grain tannin concentration after fermentation. This reduction in tannin levels is probably due to tannin inactivation by the low pH and anaerobic conditions usually present in silages (McSweeney et al., 1999) and to tannin degradation to low-molecular weight polyphenols (Kondo et al., 2004). It was also found that ensiling reduced grain tannin concentrations, but this effect was not so evident in leaf and stem tannin levels (Cummins, 1971). Nevertheless, few studies on effects of sorghum tannin on the ensiling process have measured tannin levels in plant parts. The CP concentration was higher in the silage without PEG, whereas NH3 –N levels were lower, which is probably due to lower CP degradation in the presence of tannins. These findings are consistent with Gonc¸alves et al. (1999). According to Albrecht and Muck (1991), NH3 –N reduction is a consequence of lower CP availability due to formation of tannin–protein complexes and inhibition of plant and/or bacterial proteolytic enzymes. In addition to proteins, tannins may also form complexes with structural carbohydrates and affect their degradation rate (Hervás et al., 2003). The lower aNDFom and ADFom concentrations in HT silages are probably due to the higher proportion of grains in the original plant. Makkar (2003) suggested that the detergent system must be used with caution to determine cell wall constituents in tanniferous forage because the tannin–protein complex may be associated with the fiber fraction of the plant, and thus be detected as a constituent of the cell wall. This may explain the higher lignin(sa) and ADIN/N levels in the HT silage with no PEG addition. Tannin effects when

6 Table 5 Chemical composition of the silages produced with sorghum hybrids (low and high-tannin) with urea or concentrate addition at silo opening (0 day) and after exposure to air (10 days). LT sorghum

HT sorghum

SEM

Urea

Concentrate

Urea

Concentrate

DM (g/kg) 0 10

382.5 408.0

570.1 727.5

410.1 400.6

572.9 710.7

2.80

CP (g/kg DM) 0 10

121.5a 106.3b

155.9b 213.8a

105.5b 131.2a

154.7b 205.9a

aNDFom (g/kg DM) 0 610.6 10 555.4

395.1 373.1

557.3 525.6

ADFom (g/kg DM) 0 313.9a 10 285.8b

176.3a 171.5b

302.1 302.4

38.7b 49.6a

71.8b 186.4a

ADIN/N (g/kg N) 0 10

56.9b 128.8a

P S×C

S×T

C×T

S×C×T

Conc.

Time

0.849

<0.001

<0.001

0.296

0.104

<0.001c

0.636

0.82

0.992

<0.001

<0.001

0.086

0.003

<0.001

<0.001

400.3 368.3

1.93

<0.001

<0.001

<0.001

<0.001d

0.485

0.102

0.095

182.0 175.1

1.32

0.199

<0.001

0.002

0.666

0.024

0.146

0.010

1.03

<0.001

<0.001

<0.001

0.086

<0.001

<0.001

0.040

39.0b 87.3a

Sorghum

LT = low-tannin sorghum silage; HT = high-tannin sorghum silage. DM = dry matter; CP = crude protein; aNDFom = neutral detergent fiber; ADFom = acid detergent fiber; ADIN/N = acid detergent insoluble N (g/kg N). a,b Means followed by different letters in the same column within response parameter differ (P<0.05). c The effect of concentrate was greater (P<0.05) at 10 versus 0 days. d The effect of concentrate was greater (P<0.05) within LT sorghum.

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Time (days)

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PEG was not added were probably overestimated because the tannin–protein complex was associated with the cell wall. 4.2. Experiment 2 Despite the low tannin levels in the silages after fermentation (i.e., 1 g/kg DM), the presence of tannins in the forages increased the time required to reach maximum temperature. However, both HT and LT silages had high temperatures, but different times to reach maximum values, suggesting that tannins may not have been efficient in reducing aerobic degradation caused by the high temperatures in the present study. There was a marked increase in mass temperature due to exposure to air that resulted in higher ADIN/N ratios in all treatments. The Maillard reaction is characterized by the formation of complexes of amino groups with carbohydrates (particularly between hemicellulose and soluble carbohydrates) when the temperature of the material increases (Van Soest, 1994). Such compounds are usually recovered in the analysis of residual N bound to the cell wall (Nelson and Bozich, 1996), and therefore may explain the increase in silage ADIN/N ratios after heating. However, the observed increase in ADIN/N levels due exposure to air was more evident in the treatments where urea was added, compared to concentrate supplementation, although the heating of silages with urea was less intense compared to the heating of silages supplemented with concentrate. Since ADIN/N level is expressed relative to N in the sample, it is possible that it may have been diluted by the higher CP level of the treatments with concentrate addition. The increase in CP level during the aerobic stability evaluation period mainly results from the presence of a higher number of microorganisms, as detected by mold growth in the ensiled mass. An alternative explanation for increase in ADIN/N levels due exposure to air is that urea may have increased the levels of ammonia in the silage and therefore increased pH (Van Soest, 1994). Previous studies have shown that the Maillard reaction may be intensified by higher pH (Nelson and Bozich, 1996), thereby increasing ADIN/N in silages containing urea, as observed in the present study. 5. Conclusions The silages produced with IF305 and BR700 sorghum hybrids had high nutritional values independent of tannin levels. Considering the high silage temperatures during the evaluation of aerobic stability, it seems that the tannin levels in the present study were not effective in protecting the silages against aerobic degradation. Despite the longer intervals required to achieve maximum temperature in the presence of tannins, temperatures were very high overall and negatively affected the nutritional value of silages, particularly when concentrate was supplemented to the diet. Acknowledgements The authors thank Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the scholarships, and to Dow Agrosciences for providing material to sorghum cultures. T.T. Berchielli was granted a CNPq productivity fellowship. References Adesogan, A.T., Salawu, M.B., 2002. The effect of different additives on the fermentation quality, aerobic stability and in vitro digestibility of pea/wheat bi-crop silages containing contrasting pea to wheat ratios. Grass Forage Sci. 57, 25–32. Albrecht, K.A., Muck, R.E., 1991. Proteolysis in ensiled legumes that vary in tannin concentration. Crop Sci. 31, 464–469. Association of Official Analytical Chemists, 1990. 15th ed., Association of Official Analytical Methods Inc., Arlington, VA, USA, p. 770. Barry, T.N., McNabb, W.C., 1999. The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants. Br. J. Nutr. 81, 263–272. Ben Salem, H., Nefzaoui, A., Ben Salem, L., 1999. Intake, digestibility, urinary excretion of purine derivates and growth by sheep given fresh, air-dried or polyethylene glycol-treated foliage of Acacia cyanophylla Lindl. Anim. Feed Sci. Technol. 78, 297–311. Ben Salem, H., Ben Salem, I., Nefzaoui, A., Ben Saïd, M.S., 2003. Effect of PEG and olive cake feed blocks supply on feed intake, digestion, and health of goats given kermes oak (Quercus coccifera L.) foliage. Anim. Feed Sci. Technol. 110, 45–59.

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