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Effect of enzyme addition to forage at ensiling on silage chemical composition and NDF degradation characteristics
Livestock Science 150 (2012) 51–58
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Effect of enzyme addition to forage at ensiling on silage chemical composition and NDF degradation characteristics M.R. Dehghani a,b, M.R. Weisbjerg a,n, T. Hvelplund a, N.B. Kristensen a a b
Department of Animal Science, Faculty of Science and Technology, Research Centre Foulum, Aarhus University, P.O. Box 50, DK-8830 Tjele, Denmark Animal Science Department, Agriculture College, University of Zabol, P.O. Box 9861335856, Zabol, Iran
a r t i c l e i n f o
abstract
Article history: Received 17 October 2011 Received in revised form 30 July 2012 Accepted 31 July 2012
The effect of different exogenous fibrolytic enzymes added to forages at ensiling was examined for effect on chemical composition and in vitro NDF degradability characteristics of the resulting silage. Maize stover and lucerne were used to study effect on chemical composition in experiment 1, and two varieties of maize stover, lucerne and grass clover were used to study NDF degradation characteristics in experiment 2. Forages were treated with enzymes (500 mg crude protein of the enzyme products/kg DM) and ensiled for 60 days in vacuum-sealed bags. Samples of forage (before ensiling) and silage were analysed for chemical composition and silages were analysed for pH and fermentation products. The in vitro NDF degradation characteristics of four forages treated with selected enzymes were measured by incubation for up to 96 h with rumen fluid. Enzymes with glucanase, b-glucanase and pectinase activity increased lactic acid and decreased butyric acid, ammonia and pH compared with control silage, and increased glucose concentration in lucerne silage. NDF concentration generally decreased due to enzyme treatment with glucanase, b-glucanase and xylanase activity and in vitro organic matter digestibility decreased in treated maize stover silage. Potential NDF degradability decreased due to enzyme treatments but not for all maize stover treatments. Treatments with combination of enzymes with glucanase, b-glucanase and pectinase activity mostly resulted in increases in fermentation products compared with treatment with individual enzymes. Enzyme mix with xylanase, glucanase and b-glucanase activities was effective for maize stover, whereas a mix containing pectinase activity was most effective for reducing pH in lucerne. Data from this study suggest that adding fibrolytic enzymes to forages at ensiling can solubilise some of the easily digestible parts of cell wall carbohydrates and thereby supply substrates for lactate fermentation. Therefore enzyme addition could be efficient to improve silage quality in forages which are difficult to ensile due to low sugar concentration and/or high protein concentration and buffer capacity. Further, treatment with the enzymes reduced the NDF concentration and thereby might increase the energy value and enzyme treatment might be beneficial to increase DM digestibility in high producing dairy cattle with short rumen retention time. & 2012 Elsevier B.V. All rights reserved.
Keywords: Enzyme aNDF degradation Maize stover Lucerne Grass clover Silage
Abbreviations: EZ, enzyme; DM, dry matter; NDF, neutral detergent fibre; iNDF, indigestible NDF; CP, crude protein; OM, organic matter; ED, effective degradability; c, fractional rate of degradation n Corresponding author. Tel.: þ45 87158046; fax: þ45 87154249. E-mail address: [email protected] (M.R. Weisbjerg). 1871-1413/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2012.07.031
1. Introduction Cell wall degrading enzymes have been added to forage at ensiling to improve fermentation and animal
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performance. Cell wall degrading enzymes should degrade cell wall components to simpler molecules, thereby providing lactic acid bacteria with more fermentable substrate (Kung et al., 2003; McDonald et al., 1991). Therefore, cellulase and hemicellulase addition to forage prior to ensiling should promote production of readily fermentable sugars from fibre hydrolysis, leading to a rapid pH decrease and improved ensiling characteristics (Fahey et al., 1993). When enzymes are added before ensiling, it in theory allows cell wall hydrolysis into fermentable substrates to enhance fermentation, but the literature is inconsistent on the effects. Only one out of eight fibrolytic enzymes improved fibre degradability when added to maize at ensiling (Eun and Beauchemin, 2007). Mandebvu et al. (1999) reported that fibrolytic enzymes increased butyric acid concentration and had no effect on in vitro or in situ DM or NDF disappearance of bermudagrass silage. Fibrolytic enzymes decreased pH of silage of mixed grass– legume compared with control (Stokes, 1992). According to Shepherd and Kung (1996) fibrolytic enzymes had no effect on fermentation acids, nitrogen and in vitro NDF digestion in corn silage. Colombatto et al. (2004) reported that fibrolytic enzymes reduced pH and NDF, and increased rate of degradation of organic matter in corn silage. When fibrolytic enzymes were added to lucerne at ensiling, lactic acid and glucose increased and ammonia N, NDF and ADF concentration decreased whereas in vitro digestion of NDF was unaffected (Nadeau et al., 2000a; Shepherd et al., 1995), and in the studies of Kozelov et al. (2008) pH and NDF were unaffected whereas early lactic acid production increased. When fibrolytic enzymes were added to forages before in vitro incubation, the easiest digestible NDF was solubilised, but the potential NDF digestibility did not increase (Moharrery et al., 2009). As the concentration and digestibility of NDF in forages determine the organic matter digestibility (Weisbjerg et al., 2004), an eventual effect of enzyme addition on the NDF fraction could change the energy value of the resultant silage. The hypothesis in this experiment was that treatment of forages with fibrolytic enzymes improves silage fermentation quality due to higher substrate availability and might improve NDF digestibility. The objective was to examine the effect of different exogenous enzymes with mainly cellulolytic and hemicellulytic activities when added to forages at ensiling on chemical composition, and in vitro NDF degradability characteristics of the silage.
2. Materials and methods 2.1. Forages and harvest Experiment 1, chemical composition: Maize stover was harvested on December 16th, 2009, and lucerne was harvested on July 1st, on first cut in 2010. Experiment 2, NDF degradation: Maize stover from two different varieties of maize was harvested on October 31st, 2008. Lucerne and grass-clover were harvested in a late fourth cut at the same time in Foulum (Denmark). Forages from both experiments were chopped to approx. 20 mm length with a forage chopper before ensiling. All forages were grown at Foulum (Denmark). Chemical composition of fresh forages in experiments 1 and 2 is shown in Table 1. 2.2. Ensiling and enzyme treatment The chopped forages were ensiled in vacuum-sealed bags after thoroughly mixing and treatment with enzymes. One kilogram of fresh forage (900 g for lucerne in experiment 1 was weighed out per vacuum bag for ensiling, in experiment 2 one bag per feed and treatment, in experiment 1 in duplicate. Five hundred milligram crude protein of the enzyme product was added per kg forage DM. When enzyme mixes were used, 500 mg crude protein from each enzyme product was added. Before addition, the enzyme was dissolved in 50 ml water, and 50 ml pure water was used for control. The mix was sprayed directly on the forages in the bags during mixing. Thereafter, bags were evacuated and heat sealed and stored for ensiling at 19 1C for 60 days in dark. After 60 days, ensiling process was stopped by freezing bags. Experiment 1 included 13 enzyme treatments and control. Experiment 2 included seven enzyme treatments and control, however only the three treatments with the most pronounced effect on reduction in NDF concentration were used for NDF degradation studies and therefore reported here. Chemical composition of silages from all eight treatments has been published by Hvelplund et al. (in press). The enzyme types, specifications and activities are presented in Table 2. Enzymes were provided from Novozymes, 2860 Bagsværd, Denmark. 2.3. Sampling and chemical analysis After thawing, silage juice was extracted from a sample of each silage by pressure without water addition. pH was measured immediately in the juice using a glass
Table 1 Chemical composition and in vitro organic matter (OM) digestibility of the forages.
DM (%) % of DM Crude ash Crude protein NDF In vitro OM digestibility (% of OM)
Maize stover (Variety NK Bull) Exp. 1
Lucerne Exp. 1
Maize stover (Variety Claxxon) Exp. 2
Maize stover (Variety NK Bull) Exp. 2
Grass clover Exp. 2
Lucerne Exp. 2
27.6
16.6
20.4
25.2
17.5
17.6
3.0 4.0 74.1 48.4
9.6 26 34.5 67.9
5.0 7.7 68.3 51.6
5.3 8.8 60.5 57.2
9.9 16.6 37.8 77.4
10.6 28.6 25.9 73.6
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Table 2 Activity in used enzyme products. Enzyme
Enzyme activity Glucanase
EZ1 EZ2 EZ3 EZ4 EZ12 EZ13 EZ14 EZ15
Multicomponent cellulase Multicomponent product w/b-glucosidase Multicomponent glucanase, pectinase Multicomponent xylanase Xylanase Glucanase Multicomponent cellulase Starch degrading enzyme
b-glucosidase
b-glucanase
Pectinase
X
X
Xylanase
Amylase
X X X
X X X X
electrode. An 8-mL subsample of silage juice was stabilized by adding 2 mL of 25% metaphosphoric acid and stored at 20 1C until analysis for glucose and L-lactic acid using the YSI analyser (YSI 7100, YSI Inc., Yellow Springs, OH). VFA (acetic, propionic, butyric, isobutyic, isovaleric, valeric, and capronic acid) were measured by gas chromatography (Kristensen et al., 1996). NH3 was determined using a Cobas Mira Autoanalyzer (Triolab A/S, Brøndby, Denmark) and a kit based on glutamate dehydrogenase (AM 1015, Randox Laboratories Ltd., Crumlin, UK). Dry matter concentration in forages and silages were determined at 60 1C. Dried samples were milled on 1 mm screen for chemical analysis. Ash was determined by combustion at 525 1C. Ash free NDF was analysed according to Mertens (2002) using heat stable amylase, crude protein as 6.25xN using the Dumas method according to Hansen (1989) and in vitro organic matter (OM) digestibility determined according to Tilley and Terry (1963).
2.4. In vitro NDF digestion In vitro NDF degradation profiles were determined on 16 silage samples from experiment 2, from four forages (Maize stover var. Claxxon, Maize stover var. Bull, grassclover and lucerne) combined with four enzyme treatments (EZ1, EZ3, EZ1 þEZ3 and untreated). Rumen fluid was collected from three rumen cannulated dry Danish Holstein cows and filtered through two layers of surgical gauze to thermo flasks for transport to laboratory. Rumen fluid was mixed with a buffer solution in a ratio 1:5 (v/v). Buffer composition was: 9.8 g NaHCO3, 4.62 g Na2HPO4, 0.46 g NaCl, 0.57 g KCl, 0.04 g CaCl2 and 0.69 g MgCl2 per 1000 ml. Sixty millilitre CO2 saturated rumen fluid–buffer mixture was added to glass tubes containing 0.5 g of sample and incubated at 39 1C in a water bath. Incubation times were: 0, 2, 4, 8, 24, 48 and 96 h. A blank sample containing only mixture of rumen fluid and buffer solution was included for each incubation time. Three repetitions were performed in three separate weeks; each repetition consisted of 16 samples seven incubation times, in total 112 tubes per repetition. At the end of incubation, tubes were transferred to a water bath (70 1C) to stop fermentation. Afterwards, the residues were transferred to porosity 2 filter crucibles and ash free NDF residues were determined according to Mertens (2002).
X X
2.5. Degradation parameter estimation NDF degradation parameters were estimated using the exponential equation P¼b(1e ct) (McDonald, 1981), where P is proportion disappeared at time t, b is the potentially degradable NDF, c is the fractional rate of degradation (/h) and t is the incubation time (h). Effective degradability of NDF was estimated as b [c/ (cþ0.02)] where 0.02/h is assumed fractional rate of passage. 2.6. Statistical analyses Data were analysed using the general linear model (GLM) procedure of SAS (2003). Data from experiment 1 were analysed within feed (maize stover, lucerne) with a model including effect of treatment. NDF degradation data (experiment 2) were analysed within feed with a model that included week and enzyme treatment. Data for NDF residues used for Fig. 1 were analysed within feed and incubation time with a model including enzyme treatment. Pairwise comparison to control treatment was made using PDIFF when overall treatment effect was significant (Po0.05). 3. Results 3.1. Experiment 1 Chemical composition of silage of maize stover and lucerne (experiment 1) are shown in Table 3. Dry matter and NDF concentration decreased for several of the enzyme treatments for maize stover. Enzyme treatment decreased in vitro organic matter digestibility for several enzyme treatments for maize stover compared with control. Dry matter concentration of lucerne treated with NZ4, EZ1þ EZ3 and EZ12þ EZ13 increased compared with control and NDF decreased for some enzyme treatments compared with control, especially EZ1þEZ3. Fermentation products in silage of maize stover and lucerne with and without enzyme treatment are presented in Table 4 for experiment 1. Treatment with EZ12þEZ13 increased acetic and propionic acid concentration in maize
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Fig. 1. Residues of NDF (y-axis) for silage at different times of incubation in vitro. Symbols (’, ’, K and ~) show control, EZ1, EZ3 and EZ1 þ EZ3 treatments, respectively. Residue from 0.5 g sample weigh out. Following treatments differ (P o 0.05) from control within feed and incubation time: Bull maize stover silage, 0 h all, 24 h EZ1 þEZ3, 96 h EZ1 þEZ3; Grass-clover silage, 0 h all, 4 h all, 8 h all, 96 h EZ1 and EZ1 þEZ3; Lucerne silage, 0 h all, 2 h all, 4 h all, 8 h all, 96 h EZ3. Table 3 Chemical composition of silage of maize stover and lucerne with and without enzyme treatment during ensiling (N¼ 2). Enzyme
DM (%) Maize stover
EZ1 EZ2 EZ3 EZ4 EZ12 EZ13 EZ14 EZ15 EZ1 þEZ3 EZ12þEZ13 EZ12þEZ14 EZ13þEZ14 EZ12þEZ13 þEZ14 Control SEM P treatment
26.1 27.3 27.4 27.5 25.8 26.1 24.7nn 26.6 25nn 25.4n 25.1nn 25.2nn 26.3 26.9 0.4 0.003
Ash (% DM) Lucerne 14.9 14.5 15.2 16.0nn 15.0 14.5 14.8 14.4 15.7n 14.7nn 14.2 16.2nn 14.4 14.6 0.7 0.01
Maize stover nn
3.5 3.6nn 3.5nn 3.2 3.3 3.4n 3.3 3.6nn 3.4nn 3.4n 3.5nn 3.3 3.4n 3.1 0.1 0.007
NDF (% DM) Lucerne
Maize stover
10.8 11.1 10.8 10.6 11.1 10.7 11.0 10.8 10.4 10.7 11.0 11.0 11.0 10.1 0.3 0.9
n Within a column, means followed by star differ from control, nPo 0.05; SEM: standard error of Ls means.
stover compared with control (P¼0.006 and P¼0.01, respectively). Ammonia concentration decreased in maize stover for treatments EZ3, EZ14, EZ15, EZ1þEZ3, EZ12þEZ14, EZ13þ EZ14 and EZ12þEZ13þEZ14 compared with control (P¼ 0.03). For maize stover, some enzyme treatments decreased glucose concentration, but EZ12þEZ14, EZ13þEZ14 and EZ12þEZ13þEZ14 treatments increased glucose concentrations compared with control (Pr0.0001) (Table 4). Enzyme treatments EZ1, EZ2, EZ3, EZ4, EZ12 and EZ13
74.4 75.7 74.9 74.6 74.5 75.9n 74.3 75.6 72.7 71.8nn 69.6nn 71.1nn 71.0nn 74 0.6 r 0.0001
Lucerne nn
29.5 34.2 30.9n 32.4 34.4 32.9 33.3 30.8n 29.0nn 33.1 32.1 35.2 32.1 35.3 1.4 0.08
in vitro OM digestibility (%)
Crude protein (% DM)
Maize stover
Lucerne
Maize stover
64.0 63.2 63.0 63.6 63.0 64.6 63.3 63.8 65.0 66.5 65.8 64.8 65.7 65.2 1.3 0.6
4.0 4.3 4.3 3.7n 3.9 3.9 3.8 3.7n 4.2 4.3 4.4nn 4.2 4.3 4.0 0.1 0.002
nn
43.0 42.1nn 42.8nn 44.2 45.0 45.4 43.2n 46.1 44.5 45.9 46.1 44.9 45.8 45.0 0.5 0.0005
P o 0.01.
nn
increased lactic acid concentration (Po0.01), and enzyme treatments generally decreased pH compared with control. For lucerne, treatment with enzyme decreased acetic acid concentration in EZ1, EZ3, EZ4, EZ14, EZ1þEZ3, EZ12þEZ14, EZ12þ EZ13 compared with control, especially with EZ1þ EZ3 treatment (P¼0.01) (Table 4). Several enzyme treatments decreased propionic acid concentration compared with control, especially for EZ3 (Pr0.0001). Treatment of lucerne with EZ3, EZ4, EZ1þEZ3, EZ13þEZ14 and EZ12þEZ13þEZ14 decreased butyric acid concentration, especially for EZ12þ EZ13 (P¼0.02). Enzyme treatment
4.2nn 4.7 4.1nn 4.4nn 4.7 4.5n 4.3nn 4.6 3.9nn 4.4nn 4.2nn 4.2nn 4.1nn 4.7 0.04 r 0.0001 3.9nn 3.9 3.9nn 3.8nn 3.7nn 3.8nn 3.8nn 3.9nn 3.8nn 3.9nn 3.9nn 3.9 3.9n 4 0.02 r 0.0001 3.5nn 4.3 2.7nn 3.1nn 4.3 3.7nn 3.9 4.2 2.5nn 3.3nn 2.6nn 3.0nn 2.7nn 4.1 0.3 0.0004 0.37 0.43 0.35n 0.43 0.41 0.43 0.35n 0.32nn 0.33nn 0.4 0.33nn 0.34n 0.34n 0.43 0.02 0.03 5.8 6.8 4.9n 1.8nn 12.8 9.5 7 10.7 7.9nn 1.5nn 2.8 9.5nn 2.6nn 11 2.6 0.02
P o0.01. nn n Within a column, means followed by star differ from control, nP o 0.05; SEM: standard error of Ls means.
11.33 9.78nn 10.17nn 11.10nn 12.45nn 11.67nn 9.23 8.65 8.64 9.34 9.34 9.02 8.53 7.94 0.48 0.0002 0.6 0 0.7nn 0.3 0 0.4 1.0nn 0.2 1.2nn 1.3nn 1.0nn 0.4nn 1.3nn 0.2 0.2 r 0.0001 EZ1 1.43 EZ2 1.59 EZ3 2.06 EZ4 2.03 EZ12 1.12nn EZ13 1.01nn EZ14 1.45n EZ15 1.7 EZ1 þ EZ3 2.07 EZ12 þ EZ13 2.6 EZ12 þ EZ14 3.04n EZ13 þ EZ14 2.98n EZ12 þ EZ13 þ EZ14 3.17nn Control 2.16 SEM 0.22 P treatment r 0.0001
Maize stover Lucerne Maize stover Lucerne Lucerne Maize stover
0.2 0 0 0.1 0 0.1 0.1 0.3 0 0.3 0 0.1 0.2 0.3 0.1 0.6 295 621 87nn 281n 691n 390 421 546 141nn 287nn 171n 250nn 139nn 451 73 r 0.0001 60.4 38.6 66.8nn 51.5 35.6 43 55.8 41.3 72.0nn 57.5 55.6 45.8 66.3nn 41.9 5.5 0.003
2.49 2.18 1.73 1.69 1.66 1.55 1.58 1.42 2.92n 4.91nn 1.68 1.57 1.55 1.37 0.5 0.006
4.1 5.2 4.2nn 3.8nn 5.7 5.1 4.6n 5.1 3.7nn 4.3nn 4.8n 4 4.8 5 0.4 0.01
17 6 8 10 19 22 22 15 15 36n 5 8 5 11 5 0.01
n
Lucerne Maize stover
generally decreased ammonia concentration compared with control, most pronounced for EZ1þEZ3 (P¼0.0004). Glucose concentration increased for several treatments and most pronounced for EZ12þEZ13 and EZ12þEZ13þEZ14 (Pr0.0001). Lactic acid concentrations increased with several enzyme treatments compared with control, especially EZ1þEZ3 increased lactic acid concentration. Most enzyme treatments decreased pH significantly compared with control (Pr0.0001), especially the EZ1þ EZ3 treatment.
nn
Lucerne Maize stover Lucerne
n nn
Maize stover Maize stover Lucerne
55
3.2. Experiment 2
n
L-Lactic acid (g/kg of DM) Acetic acid (g/kg of DM) Propionic acid (mg/kg of DM) Butyric acid (mg/kg of DM) Glucose (g/kg of DM) Enzyme
Table 4 Fermentation products of maize stover and lucerne treated with enzymes (N ¼ 2).
NH3 (g/kg of DM)
pH
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Effect of enzyme treatments during ensiling on NDF digestion kinetics of silages is presented in Fig. 1, and enzyme treatments reduced NDF residues, but only had slight although in some cases significant effect on the residues after 96 h incubation. The potential degradability (Table 5) of NDF ranged from 0.20 to 0.73. Potential degradability decreased significantly for EZ1 and EZ1þ EZ3 treatments for maize stover (Claxxon) and EZ1þ EZ3 for maize stover (Bull). The potential degradability decreased for all enzyme treatments in grass clover compared with control. For lucerne silage the potential degradability of NDF showed the highest variation among treatments (from 0.56 to 0.73). Rate of degradation increased considerably for EZ3 and EZ1þEZ3 compared with control for maize stover (Bull) and resulted in increased effective degradability of NDF. For most other enzyme treatments effective degradability decreased compared with control. Based on the NDF concentrations in the silages and the effective degradabilities found, effective undegradable NDF was calculated as NDF (1 effective degradability of NDF) (Table 5). Effectively undegradable NDF was reduced by most enzyme treatments, and especially the combined EZ1 þEZ3 treatment reduced effectively undegradable NDF except for maize stover Claxxon. 4. Discussion 4.1. Experiment 1 Combination treatment with mixes of enzymes mostly resulted in larger effects compared with individual enzyme components, which might be due to synergistic effects or to the higher combined enzyme dose. Some enzyme treatments increased lactic acid concentration and decreased pH, and most pronounced for EZ12 for maize stover. The main activity of EZ12 was xylanase activity (Table 2). The combination of EZ12 þEZ14 was the most effective for increasing glucose and decreasing NDF concentration in maize stover. The combination of EZ1 and EZ3 (EZ1þ EZ3) with glucanase, b-glucanase and pectinase activity was most efficient in increasing fermentation products and in decreasing NDF concentration in lucerne silage. This combination of enzymes increased glucose and lactic acid and decreased pH and NDF and had the most pronounced effect compared with the other enzyme treatments. This is in accordance with Tengerdy et al. (1991) who found a cocktail of cellulase, hemicellulase and pectinase most enhancive in ensiling lucerne.
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Table 5 NDF concentration and degradation kinetics of maize stover, grass clover and lucerne treated with selected enzymes (N ¼3). Potential degradability
ca (/h)
Feed
Treatment
NDF concentration (% DM)
Maize stover (Claxxon)
EZ1 EZ3 EZ1 þEZ3 Control
63.9 65.0 63.5 68.3
0.33n 0.38 0.32nn 0.43 0.02
0.028 0.029 0.027 0.028 0.004
0.19nn 0.23 0.17nn 0.25 0.007
51.7 50.3 52.8 51.4 0.5
EZ1 EZ3 EZ1 þEZ3 Control
52.1 56.2 52.0 56.0
0.45 0.44 0.40 0.48 0.03
0.0220 0.041n 0.035n 0.016 0.005
0.22 0.28n 0.25n 0.22 0.01
40.6nn 40.3nn 38.9nn 43.9 0.6
EZ1 EZ3 EZ1 þEZ3 Control
36.7 35.2 30.8 42.2
0.35nn 0.33nn 0.20nn 0.44 0.01
0.038 0.017n 0.042 0.048 0.004
0.23nn 0.15nn 0.13nn 0.31 0.01
28.4 29.9 26.8nn 29.2 0.4
EZ1 EZ3 EZ1 þEZ3 Control
18.5 18.7 15.3 25.0
0.65 0.73 0.56n 0.70 0.03
0.025 0.025 0.027 0.027 0.003
0.36n 0.40 0.30nn 0.40 0.01
11.8nn 11.2nn 10.7nn 15.0 0.2
SEM Maize stover (Bull)
SEM Grass clover
SEM Lucerne
SEM P
Feed Treatment Feed Treatment
r 0.0001 r 0.0001 0.03
0.2 0.5 0.04
Effective degradabilityb
r 0.0001 r 0.0001 r 0.0001
Effective undegradable NDFc (% DM)
r 0.0001 r 0.0001 r 0.0001
n Within a column and feed, means followed by star differ from control, nPo 0.05; nnP o0.01. SEM: standard error of Ls means. a Fractional rate of degradation. b Effective degradability calculated using 0.02/h passage rate. c Effective undegradable NDF calculated as NDF (% DM) (1 effective degradability).
Enzyme treatment of maize stover for silage decreased DM concentration compared with control probably due to a loss of dry matter during fermentation process and a higher production of volatile fermentation products, which evaporate during the drying process. Several of the fibrolytic enzyme treatments were effective in degrading cell wall carbohydrates (NDF) in the tested forages and therefore reduced NDF in the treated silages. NDF concentration generally decreased compared with control due to enzyme treatment for maize stover and lucerne, indicating that enzyme treatments were effective in solubilising NDF. Similar effects were found by Higginbotham et al. (1994), Shepherd and Kung (1996), Shepherd et al. (1995) and Spoelstra et al. (1992), however Mandebvu et al. (1999) and Kozelov et al. (2008) found that fibrolytic enzyme had no effect on cell wall concentration of bermudagrass and lucerne silage, respectively. This contradiction may be due to forage and enzyme type and level in these experiments. Despite the effect on NDF concentration, the digestibility of organic matter (in vitro) was not affected by enzyme treatment for lucerne and even decreased for some enzyme treatments for maize stover. That OM digestibility did not increase despite a reduction in NDF concentration which was probably caused by a solubilisation of only the easily digestible NDF part, as discussed below, and therefore OM digestibility was unaffected. That OM digestibility even decreased for some enzyme treatments of maize stover might be due to loss of fermentation products during the drying process. Lack of increase in OM digestibility despite
considerable reductions in NDF concentration when using enzymes as additive at ensiling is in accordance with the results of Nadeau et al. (2000a) and Van Vuuren et al. (1989). Fibrolytic enzymes generally increased L-lactic acid concentration in silage compared with control. This is probably due to a release of soluble and fermentable carbohydrates by fibrolytic enzyme degradation of NDF, and this supply of substrate increased the L-lactic acid production by the lactic acid bacteria. This is in accordance with Colombatto et al. (2004) who found that lactic acid concentration increased by enzyme treatment in corn silage, whereas Mandebvu et al. (1999) and Shepherd and Kung (1996) found no effect of fibrolytic enzymes on lactic acid concentration in bermudagrass silage and corn silage, respectively. The L-lactic acid concentrations found for some lucerne treatments were high, probably due to an abundant supply of substrate from the pectin degrading enzymes, combined with low DM concentrations which has allowed for an extended fermentation. Large variations in lactic acid and pH in lucerne silage were imposed by the enzyme treatments, and the lowest pH found for the enzyme treatment including pectinase was 0.8 pH units lower than the untreated control. Reduced pH in silage treated with enzymes is in accordance with results from Colombatto et al. (2004), Nadeau et al. (2000b), Stokes (1992) and Zahiroddini et al. (2004) who observed decreased pH in silage of corn, orchardgrass, mix of grass–legume, and barley, respectively, when fibrolytic enzymes were used for ensiling.
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Concentration of acetic acid was lower in several enzyme treated lucerne silages than in control. Acetic/ lactic acid ratio decreased in enzyme treated lucerne silage compared with control; whereby treatment with fibrolytic enzymes seems to result in a more homofermentative ensiling process. Nadeau et al. (2000b) reported more heterofermentic ensiling for lucerne silage compared to orchardgrass due to higher buffering capacity, but also that enzyme induced reduction in acetic acid concentration was more pronounced for lucerne than for ochardgrass consistent to this study. However, in maize stover silage, enzyme treatment increased acetic acid concentration, most pronounced for EZ12þEZ13 combining glucanase and xylanase, as xylose mainly is fermented to acetic acid (Fred et al., 1919). Several of the enzyme tested showed an improved fermentation resulting in decreased butyric acid concentration in both maize stover and lucerne silage compared with control. Enzyme treatment might increase glucose concentration as a result of the NDF degradation. Although the released glucose was the driver for the enhanced fermentation to especially lactic acid, not all released glucose was fermented and glucose concentration in the silage increased for some treatments. Shepherd et al. (1995) and Shepherd and Kung (1996) also reported that glucose increased in lucerne silage and corn silage, respectively, due to treatment with fibrolytic enzymes. The low pH in some of the treated silage seemed to prevent proteolysis and decrease NH3 concentration. Enzyme induced enhanced fermentation is beneficial in reducing protein degradation during ensiling and is in accordance with Zahiroddini et al. (2004) for barley silage and Nadeau et al. (2000a) for ochardgrass and lucerne silage. 4.2. Experiment 2 Treatment with fibrolytic enzyme could degrade the most easily digestible parts of NDF during ensiling and thereby reduce the NDF concentration in silage DM. NDF concentration was considerably lower in enzyme treated silages compared with control for the four silages selected for NDF degradation studies. Enzyme treatment decreased the potential degradability of silages compared with control, which is in accordance to the findings by Moharrery et al. (2009) that enzymes degrade the easiest degradable NDF whereas the concentration of indigestible NDF seems to keep constant in DM but increase as a proportion of NDF. When the digestible fraction of NDF decreased due to the effect of enzymes on cell wall carbohydrates, the indigestible NDF in total NDF increased (Fig. 1). This is in contrast to Stokes (1992) and Shepherd and Kung (1996) who found that enzyme treatment at ensiling increased in vitro DM digestion and had no effect on in vitro NDF digestion of grass–legume mix and corn silage, respectively, however in accordance with results from Nadeau et al. (1996) who also found that enzyme addition at ensiling especially solubilised the easily digestible fraction of NDF. The amount of iNDF to DM was approximately constant in the present study and total degradability of NDF decreased. Although effective
57
NDF degradability decreased except for maize stover (Bull) treatment EZ3 and EZ1 þEZ, the decreased NDF concentration can result in higher availability of nutrients in an in vivo situation. Effective undegradable NDF in per cent of DM was generally reduced by enzyme treatments, indicating that although no clear reduction was seen in indigestible NDF, effective undegradable NDF can be reduced and thereby an effective enzyme treatment can be expected to increase the concentration of cell soluble substances and reduce the amount of NDF and thereby increase DM digestibility in high producing dairy cattle with short rumen retention time. 5. Conclusion Addition of fibrolytic enzymes before ensiling increased lactic acid and glucose and decreased ammonia and butyric acid concentrations and decreased pH in silages. Data from this study further suggest that adding fibrolytic enzymes to forage at ensiling can solubilise some of the easy digestible parts of cell wall carbohydrates and thereby supply substrates for lactate fermentation. Therefore enzyme addition could be efficient to improve silage quality in forages which are difficult to ensile due to low sugar concentration and/or high protein concentration and buffer capacity. Enzyme treatment at ensiling with the enzymes used in the present study did not increase in vitro organic matter digestibility, but due to the reduced concentration of NDF, DM digestibility can be expected to increase in high producing dairy cattle with short rumen retention time. Conflicts of interest statement No conflicts of interest exits.
Acknowledgements We thank Mr. Torkild Jakobsen, Mrs. Inger Østergaard and Mrs. Anne Krustrup for help with the experimental work. The project was partly funded through the FFU (DANIDA) project ‘Income generation through market access and improved feed utilisation-production of quality beef and goat meat (IGMAFU-meat)’. Novozymes is acknowledged for supply of enzymes. References Colombatto, D., Mould, F.L., Bhat, M.K., Phipps, R.H., Owen, E., 2004. In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage. I. Effects of ensiling temperature, enzyme source and addition level. Anim. Feed Sci. Technol. 111, 111–128. Eun, J.S., Beauchemin, K.A., 2007. Assessment of the efficacy of varying experimental exogenous fibrolytic enzymes using in vitro fermentation characteristics. Anim. Feed Sci. Technol. 132, 298–315. Fahey Jr., G.C., Titgemeyer, E.C., Atwell, D.G., 1993. Postharvest treatment of fibrous feedstuffs to improve their nutritive value. In: Jung, H.G., Buxton, D.R., Hatfield, R.D., Ralph, J. (Eds.), Forage Cell Wall Structure and Digestibility, ASA, CSSA, SSSA, Madison, WI, pp. 715–766. Fred, E.B., Peterson, W.H., Davenport, A., 1919. Acid fermentation of xylose. J. Biol. Chem. 39, 347–384. Hansen, B., 1989. Determination of nitrogen as elementary N, an alternative to Kjeldahl. Acta Agric. Scand. 39, 113–118.
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Higginbotham, G.E., Mueller, S.C., Collar, C.A., Schultz, T.A., 1994. Effects of an enzyme addition on corn silage fermentation. Prof. Anim. Sci. 10, 163–168. Hvelplund, T., Weisbjerg, M.R., Hindrichsen, I.K., Madsen, J. The effect of adding exogenous enzymes at ensiling on nutrient availability in different forages. In: Proceedings of the 33th Annual Scientific Conference. Society of Animal Production, Tanzania. September 2009, in press. Kozelov, L.K., Lliev, F., Hristov, A.N., Zaman, S., McAllister, T.A., 2008. Effect of fibrolytic enzyme and an inoculant on in vitro degradability and gas production of low dry matter alfalfa silage. J. Sci. Food Agric. 88, 2568–2575. Kristensen, N.B., Danfær, A., Tetens, V., Agergaard, N., 1996. Portal recovery of intraruminally infused short-chain fatty acids in sheep. Acta Agric. Scand. Anim. Sci. 46, 26–38. Kung Jr., L., Stokes, M.R., Lin, C.J., 2003. Silage additives. In: Buxton, D.R., Muck, R.E., Harrison, J.H. (Eds.), Silage Science and Technology. Agronomy Monograph, 42. , ASA, CSSA, and SSSA, Madison, WI, pp. 305–360. Mandebvu, P., West, J.W., Froetschel, M.A., Hatfield, R.D., Gates, R.N., Hill, G.M., 1999. Effect of enzyme or microbial treatments of bermudagrass forages before ensiling on cell wall composition, end products of silage fermentation and in situ digestion kinetics. Anim. Feed Sci. Technol. 77, 317–329. McDonald, I., 1981. A revised method for the estimation of protein degradability in the rumen. J. Agric. Sci.(Cambridge) 96, 251–252. McDonald, P., Henderson, A.R., Heron, S.J.E., 1991. The Biochemistry of Silage, 2nd ed. Chalcombe Publications, Marlow, Bucks, UK. Mertens, D.R., 2002. Gravimetric determination of amylase-treated neutral detergent fiber in feeds with refluxing in beakers or crucibles: collaborative study. J. AOAC Intern. 85, 1217–1240. Moharrery, A., Hvelplund, T., Weisbjerg, M.R., 2009. Effect of forage type, harvesting time and exogenouse enzyme application on degradation characteristics measured using in vitro technique. Anim. Feed Sci. Technol. 153, 178–192. Nadeau, E.M.G., Buxton, D.R., Lindgren, E., Lingvall, P., 1996. Kinetics of cell-wall digestion of ochardgrass and alfalfa silage treated with cellulase and formic acid. J. Dairy Sci. 79, 2207–2216.
Nadeau, E.M., Buxton, D.R., Russell, J.R., Allison, M.J., Young, J.W., 2000a. Enzyme, bacterial inoculant, and formic acid effects on silage composition of orchardgrass and alfalfa. J. Dairy Sci. 83, 1487–1502. Nadeau, E.M., Russell, J.R., Buxton, D.R., 2000b. Intake, digestibility, and composition of orchardgrass and alfalfa silages treated with cellulase, inoculant and formic acid fed to lambs. J. Anim. Sci. 78, 2980–2989. SAS, 2003. In: User’s Guide: Statistics, Version 9.1edition SAS Institute, Inc., Cary, NC, USA. Shepherd, A.C., Kung Jr., L., 1996. Effects of an enzyme additive on composition of corn silage ensiled at various stages of maturity. J. Dairy Sci. 79, 1767–1773. Shepherd, A.C., Maslanka, M., Quinn, D., Kung Jr., L., 1995. Additives containing bacteria and enzymes for alfalfa silage. J. Dairy Sci. 78, 565–572. Spoelstra, S.F., Van Wikselaar, P.G., Harder, B., 1992. The effects of ensiling whole crop maize with a multi-enzyme preparation on the chemical composition of the resultant silages. J. Sci. Food Agric. 60, 223–228. Stokes, M.R., 1992. Effects of an enzyme mixture, an inoculant, and their interaction on silage fermentation and dairy production. J. Dairy Sci. 75, 764–773. Tengerdy, R.P., Weinberg, Z.G., Szakacs, G., WU, M., Linfen, J.C., Henk, L.L., Johnson, D.E., 1991. Ensiling alfalfa with additives of lactic acid bacteria and enzymes. J. Sci. Food Agric. 55, 215–228. Tilley, J.M.A., Terry, R.A., 1963. A two-stage technique for the digestion of forage crops. J. Br. Grassland Soc. 18, 104–111. Van Vuuren, A.M., Bergsma, K., Frol-Kramer, F., van Beers, J.A.C., 1989. Effects of addition of cell wall degrading enzymes on the chemical composition and the in sacco degradation of grass silage. Grass Forage Sci. 44, 223–230. Weisbjerg, M.R., Hvelplund, T., Søegaard, K., 2004. Prediction of NDF digestibility based on assumptions about true digestibility and endogenous loss of NDS. J. Anim. Feed Sci. 13 (Suppl. 1), 243–246. Zahiroddini, H., Baah, J., Absalom, W., McAllister, T.A., 2004. Effect of an inoculant and hydrolytic enzymes on fermentation and nutritive value of whole crop barley silage. Anim. Feed Sci. Technol. 117, 317–330.
Contents lists available at SciVerse ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Effect of enzyme addition to forage at ensiling on silage chemical composition and NDF degradation characteristics M.R. Dehghani a,b, M.R. Weisbjerg a,n, T. Hvelplund a, N.B. Kristensen a a b
Department of Animal Science, Faculty of Science and Technology, Research Centre Foulum, Aarhus University, P.O. Box 50, DK-8830 Tjele, Denmark Animal Science Department, Agriculture College, University of Zabol, P.O. Box 9861335856, Zabol, Iran
a r t i c l e i n f o
abstract
Article history: Received 17 October 2011 Received in revised form 30 July 2012 Accepted 31 July 2012
The effect of different exogenous fibrolytic enzymes added to forages at ensiling was examined for effect on chemical composition and in vitro NDF degradability characteristics of the resulting silage. Maize stover and lucerne were used to study effect on chemical composition in experiment 1, and two varieties of maize stover, lucerne and grass clover were used to study NDF degradation characteristics in experiment 2. Forages were treated with enzymes (500 mg crude protein of the enzyme products/kg DM) and ensiled for 60 days in vacuum-sealed bags. Samples of forage (before ensiling) and silage were analysed for chemical composition and silages were analysed for pH and fermentation products. The in vitro NDF degradation characteristics of four forages treated with selected enzymes were measured by incubation for up to 96 h with rumen fluid. Enzymes with glucanase, b-glucanase and pectinase activity increased lactic acid and decreased butyric acid, ammonia and pH compared with control silage, and increased glucose concentration in lucerne silage. NDF concentration generally decreased due to enzyme treatment with glucanase, b-glucanase and xylanase activity and in vitro organic matter digestibility decreased in treated maize stover silage. Potential NDF degradability decreased due to enzyme treatments but not for all maize stover treatments. Treatments with combination of enzymes with glucanase, b-glucanase and pectinase activity mostly resulted in increases in fermentation products compared with treatment with individual enzymes. Enzyme mix with xylanase, glucanase and b-glucanase activities was effective for maize stover, whereas a mix containing pectinase activity was most effective for reducing pH in lucerne. Data from this study suggest that adding fibrolytic enzymes to forages at ensiling can solubilise some of the easily digestible parts of cell wall carbohydrates and thereby supply substrates for lactate fermentation. Therefore enzyme addition could be efficient to improve silage quality in forages which are difficult to ensile due to low sugar concentration and/or high protein concentration and buffer capacity. Further, treatment with the enzymes reduced the NDF concentration and thereby might increase the energy value and enzyme treatment might be beneficial to increase DM digestibility in high producing dairy cattle with short rumen retention time. & 2012 Elsevier B.V. All rights reserved.
Keywords: Enzyme aNDF degradation Maize stover Lucerne Grass clover Silage
Abbreviations: EZ, enzyme; DM, dry matter; NDF, neutral detergent fibre; iNDF, indigestible NDF; CP, crude protein; OM, organic matter; ED, effective degradability; c, fractional rate of degradation n Corresponding author. Tel.: þ45 87158046; fax: þ45 87154249. E-mail address: [email protected] (M.R. Weisbjerg). 1871-1413/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2012.07.031
1. Introduction Cell wall degrading enzymes have been added to forage at ensiling to improve fermentation and animal
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performance. Cell wall degrading enzymes should degrade cell wall components to simpler molecules, thereby providing lactic acid bacteria with more fermentable substrate (Kung et al., 2003; McDonald et al., 1991). Therefore, cellulase and hemicellulase addition to forage prior to ensiling should promote production of readily fermentable sugars from fibre hydrolysis, leading to a rapid pH decrease and improved ensiling characteristics (Fahey et al., 1993). When enzymes are added before ensiling, it in theory allows cell wall hydrolysis into fermentable substrates to enhance fermentation, but the literature is inconsistent on the effects. Only one out of eight fibrolytic enzymes improved fibre degradability when added to maize at ensiling (Eun and Beauchemin, 2007). Mandebvu et al. (1999) reported that fibrolytic enzymes increased butyric acid concentration and had no effect on in vitro or in situ DM or NDF disappearance of bermudagrass silage. Fibrolytic enzymes decreased pH of silage of mixed grass– legume compared with control (Stokes, 1992). According to Shepherd and Kung (1996) fibrolytic enzymes had no effect on fermentation acids, nitrogen and in vitro NDF digestion in corn silage. Colombatto et al. (2004) reported that fibrolytic enzymes reduced pH and NDF, and increased rate of degradation of organic matter in corn silage. When fibrolytic enzymes were added to lucerne at ensiling, lactic acid and glucose increased and ammonia N, NDF and ADF concentration decreased whereas in vitro digestion of NDF was unaffected (Nadeau et al., 2000a; Shepherd et al., 1995), and in the studies of Kozelov et al. (2008) pH and NDF were unaffected whereas early lactic acid production increased. When fibrolytic enzymes were added to forages before in vitro incubation, the easiest digestible NDF was solubilised, but the potential NDF digestibility did not increase (Moharrery et al., 2009). As the concentration and digestibility of NDF in forages determine the organic matter digestibility (Weisbjerg et al., 2004), an eventual effect of enzyme addition on the NDF fraction could change the energy value of the resultant silage. The hypothesis in this experiment was that treatment of forages with fibrolytic enzymes improves silage fermentation quality due to higher substrate availability and might improve NDF digestibility. The objective was to examine the effect of different exogenous enzymes with mainly cellulolytic and hemicellulytic activities when added to forages at ensiling on chemical composition, and in vitro NDF degradability characteristics of the silage.
2. Materials and methods 2.1. Forages and harvest Experiment 1, chemical composition: Maize stover was harvested on December 16th, 2009, and lucerne was harvested on July 1st, on first cut in 2010. Experiment 2, NDF degradation: Maize stover from two different varieties of maize was harvested on October 31st, 2008. Lucerne and grass-clover were harvested in a late fourth cut at the same time in Foulum (Denmark). Forages from both experiments were chopped to approx. 20 mm length with a forage chopper before ensiling. All forages were grown at Foulum (Denmark). Chemical composition of fresh forages in experiments 1 and 2 is shown in Table 1. 2.2. Ensiling and enzyme treatment The chopped forages were ensiled in vacuum-sealed bags after thoroughly mixing and treatment with enzymes. One kilogram of fresh forage (900 g for lucerne in experiment 1 was weighed out per vacuum bag for ensiling, in experiment 2 one bag per feed and treatment, in experiment 1 in duplicate. Five hundred milligram crude protein of the enzyme product was added per kg forage DM. When enzyme mixes were used, 500 mg crude protein from each enzyme product was added. Before addition, the enzyme was dissolved in 50 ml water, and 50 ml pure water was used for control. The mix was sprayed directly on the forages in the bags during mixing. Thereafter, bags were evacuated and heat sealed and stored for ensiling at 19 1C for 60 days in dark. After 60 days, ensiling process was stopped by freezing bags. Experiment 1 included 13 enzyme treatments and control. Experiment 2 included seven enzyme treatments and control, however only the three treatments with the most pronounced effect on reduction in NDF concentration were used for NDF degradation studies and therefore reported here. Chemical composition of silages from all eight treatments has been published by Hvelplund et al. (in press). The enzyme types, specifications and activities are presented in Table 2. Enzymes were provided from Novozymes, 2860 Bagsværd, Denmark. 2.3. Sampling and chemical analysis After thawing, silage juice was extracted from a sample of each silage by pressure without water addition. pH was measured immediately in the juice using a glass
Table 1 Chemical composition and in vitro organic matter (OM) digestibility of the forages.
DM (%) % of DM Crude ash Crude protein NDF In vitro OM digestibility (% of OM)
Maize stover (Variety NK Bull) Exp. 1
Lucerne Exp. 1
Maize stover (Variety Claxxon) Exp. 2
Maize stover (Variety NK Bull) Exp. 2
Grass clover Exp. 2
Lucerne Exp. 2
27.6
16.6
20.4
25.2
17.5
17.6
3.0 4.0 74.1 48.4
9.6 26 34.5 67.9
5.0 7.7 68.3 51.6
5.3 8.8 60.5 57.2
9.9 16.6 37.8 77.4
10.6 28.6 25.9 73.6
M.R. Dehghani et al. / Livestock Science 150 (2012) 51–58
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Table 2 Activity in used enzyme products. Enzyme
Enzyme activity Glucanase
EZ1 EZ2 EZ3 EZ4 EZ12 EZ13 EZ14 EZ15
Multicomponent cellulase Multicomponent product w/b-glucosidase Multicomponent glucanase, pectinase Multicomponent xylanase Xylanase Glucanase Multicomponent cellulase Starch degrading enzyme
b-glucosidase
b-glucanase
Pectinase
X
X
Xylanase
Amylase
X X X
X X X X
electrode. An 8-mL subsample of silage juice was stabilized by adding 2 mL of 25% metaphosphoric acid and stored at 20 1C until analysis for glucose and L-lactic acid using the YSI analyser (YSI 7100, YSI Inc., Yellow Springs, OH). VFA (acetic, propionic, butyric, isobutyic, isovaleric, valeric, and capronic acid) were measured by gas chromatography (Kristensen et al., 1996). NH3 was determined using a Cobas Mira Autoanalyzer (Triolab A/S, Brøndby, Denmark) and a kit based on glutamate dehydrogenase (AM 1015, Randox Laboratories Ltd., Crumlin, UK). Dry matter concentration in forages and silages were determined at 60 1C. Dried samples were milled on 1 mm screen for chemical analysis. Ash was determined by combustion at 525 1C. Ash free NDF was analysed according to Mertens (2002) using heat stable amylase, crude protein as 6.25xN using the Dumas method according to Hansen (1989) and in vitro organic matter (OM) digestibility determined according to Tilley and Terry (1963).
2.4. In vitro NDF digestion In vitro NDF degradation profiles were determined on 16 silage samples from experiment 2, from four forages (Maize stover var. Claxxon, Maize stover var. Bull, grassclover and lucerne) combined with four enzyme treatments (EZ1, EZ3, EZ1 þEZ3 and untreated). Rumen fluid was collected from three rumen cannulated dry Danish Holstein cows and filtered through two layers of surgical gauze to thermo flasks for transport to laboratory. Rumen fluid was mixed with a buffer solution in a ratio 1:5 (v/v). Buffer composition was: 9.8 g NaHCO3, 4.62 g Na2HPO4, 0.46 g NaCl, 0.57 g KCl, 0.04 g CaCl2 and 0.69 g MgCl2 per 1000 ml. Sixty millilitre CO2 saturated rumen fluid–buffer mixture was added to glass tubes containing 0.5 g of sample and incubated at 39 1C in a water bath. Incubation times were: 0, 2, 4, 8, 24, 48 and 96 h. A blank sample containing only mixture of rumen fluid and buffer solution was included for each incubation time. Three repetitions were performed in three separate weeks; each repetition consisted of 16 samples seven incubation times, in total 112 tubes per repetition. At the end of incubation, tubes were transferred to a water bath (70 1C) to stop fermentation. Afterwards, the residues were transferred to porosity 2 filter crucibles and ash free NDF residues were determined according to Mertens (2002).
X X
2.5. Degradation parameter estimation NDF degradation parameters were estimated using the exponential equation P¼b(1e ct) (McDonald, 1981), where P is proportion disappeared at time t, b is the potentially degradable NDF, c is the fractional rate of degradation (/h) and t is the incubation time (h). Effective degradability of NDF was estimated as b [c/ (cþ0.02)] where 0.02/h is assumed fractional rate of passage. 2.6. Statistical analyses Data were analysed using the general linear model (GLM) procedure of SAS (2003). Data from experiment 1 were analysed within feed (maize stover, lucerne) with a model including effect of treatment. NDF degradation data (experiment 2) were analysed within feed with a model that included week and enzyme treatment. Data for NDF residues used for Fig. 1 were analysed within feed and incubation time with a model including enzyme treatment. Pairwise comparison to control treatment was made using PDIFF when overall treatment effect was significant (Po0.05). 3. Results 3.1. Experiment 1 Chemical composition of silage of maize stover and lucerne (experiment 1) are shown in Table 3. Dry matter and NDF concentration decreased for several of the enzyme treatments for maize stover. Enzyme treatment decreased in vitro organic matter digestibility for several enzyme treatments for maize stover compared with control. Dry matter concentration of lucerne treated with NZ4, EZ1þ EZ3 and EZ12þ EZ13 increased compared with control and NDF decreased for some enzyme treatments compared with control, especially EZ1þEZ3. Fermentation products in silage of maize stover and lucerne with and without enzyme treatment are presented in Table 4 for experiment 1. Treatment with EZ12þEZ13 increased acetic and propionic acid concentration in maize
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Fig. 1. Residues of NDF (y-axis) for silage at different times of incubation in vitro. Symbols (’, ’, K and ~) show control, EZ1, EZ3 and EZ1 þ EZ3 treatments, respectively. Residue from 0.5 g sample weigh out. Following treatments differ (P o 0.05) from control within feed and incubation time: Bull maize stover silage, 0 h all, 24 h EZ1 þEZ3, 96 h EZ1 þEZ3; Grass-clover silage, 0 h all, 4 h all, 8 h all, 96 h EZ1 and EZ1 þEZ3; Lucerne silage, 0 h all, 2 h all, 4 h all, 8 h all, 96 h EZ3. Table 3 Chemical composition of silage of maize stover and lucerne with and without enzyme treatment during ensiling (N¼ 2). Enzyme
DM (%) Maize stover
EZ1 EZ2 EZ3 EZ4 EZ12 EZ13 EZ14 EZ15 EZ1 þEZ3 EZ12þEZ13 EZ12þEZ14 EZ13þEZ14 EZ12þEZ13 þEZ14 Control SEM P treatment
26.1 27.3 27.4 27.5 25.8 26.1 24.7nn 26.6 25nn 25.4n 25.1nn 25.2nn 26.3 26.9 0.4 0.003
Ash (% DM) Lucerne 14.9 14.5 15.2 16.0nn 15.0 14.5 14.8 14.4 15.7n 14.7nn 14.2 16.2nn 14.4 14.6 0.7 0.01
Maize stover nn
3.5 3.6nn 3.5nn 3.2 3.3 3.4n 3.3 3.6nn 3.4nn 3.4n 3.5nn 3.3 3.4n 3.1 0.1 0.007
NDF (% DM) Lucerne
Maize stover
10.8 11.1 10.8 10.6 11.1 10.7 11.0 10.8 10.4 10.7 11.0 11.0 11.0 10.1 0.3 0.9
n Within a column, means followed by star differ from control, nPo 0.05; SEM: standard error of Ls means.
stover compared with control (P¼0.006 and P¼0.01, respectively). Ammonia concentration decreased in maize stover for treatments EZ3, EZ14, EZ15, EZ1þEZ3, EZ12þEZ14, EZ13þ EZ14 and EZ12þEZ13þEZ14 compared with control (P¼ 0.03). For maize stover, some enzyme treatments decreased glucose concentration, but EZ12þEZ14, EZ13þEZ14 and EZ12þEZ13þEZ14 treatments increased glucose concentrations compared with control (Pr0.0001) (Table 4). Enzyme treatments EZ1, EZ2, EZ3, EZ4, EZ12 and EZ13
74.4 75.7 74.9 74.6 74.5 75.9n 74.3 75.6 72.7 71.8nn 69.6nn 71.1nn 71.0nn 74 0.6 r 0.0001
Lucerne nn
29.5 34.2 30.9n 32.4 34.4 32.9 33.3 30.8n 29.0nn 33.1 32.1 35.2 32.1 35.3 1.4 0.08
in vitro OM digestibility (%)
Crude protein (% DM)
Maize stover
Lucerne
Maize stover
64.0 63.2 63.0 63.6 63.0 64.6 63.3 63.8 65.0 66.5 65.8 64.8 65.7 65.2 1.3 0.6
4.0 4.3 4.3 3.7n 3.9 3.9 3.8 3.7n 4.2 4.3 4.4nn 4.2 4.3 4.0 0.1 0.002
nn
43.0 42.1nn 42.8nn 44.2 45.0 45.4 43.2n 46.1 44.5 45.9 46.1 44.9 45.8 45.0 0.5 0.0005
P o 0.01.
nn
increased lactic acid concentration (Po0.01), and enzyme treatments generally decreased pH compared with control. For lucerne, treatment with enzyme decreased acetic acid concentration in EZ1, EZ3, EZ4, EZ14, EZ1þEZ3, EZ12þEZ14, EZ12þ EZ13 compared with control, especially with EZ1þ EZ3 treatment (P¼0.01) (Table 4). Several enzyme treatments decreased propionic acid concentration compared with control, especially for EZ3 (Pr0.0001). Treatment of lucerne with EZ3, EZ4, EZ1þEZ3, EZ13þEZ14 and EZ12þEZ13þEZ14 decreased butyric acid concentration, especially for EZ12þ EZ13 (P¼0.02). Enzyme treatment
4.2nn 4.7 4.1nn 4.4nn 4.7 4.5n 4.3nn 4.6 3.9nn 4.4nn 4.2nn 4.2nn 4.1nn 4.7 0.04 r 0.0001 3.9nn 3.9 3.9nn 3.8nn 3.7nn 3.8nn 3.8nn 3.9nn 3.8nn 3.9nn 3.9nn 3.9 3.9n 4 0.02 r 0.0001 3.5nn 4.3 2.7nn 3.1nn 4.3 3.7nn 3.9 4.2 2.5nn 3.3nn 2.6nn 3.0nn 2.7nn 4.1 0.3 0.0004 0.37 0.43 0.35n 0.43 0.41 0.43 0.35n 0.32nn 0.33nn 0.4 0.33nn 0.34n 0.34n 0.43 0.02 0.03 5.8 6.8 4.9n 1.8nn 12.8 9.5 7 10.7 7.9nn 1.5nn 2.8 9.5nn 2.6nn 11 2.6 0.02
P o0.01. nn n Within a column, means followed by star differ from control, nP o 0.05; SEM: standard error of Ls means.
11.33 9.78nn 10.17nn 11.10nn 12.45nn 11.67nn 9.23 8.65 8.64 9.34 9.34 9.02 8.53 7.94 0.48 0.0002 0.6 0 0.7nn 0.3 0 0.4 1.0nn 0.2 1.2nn 1.3nn 1.0nn 0.4nn 1.3nn 0.2 0.2 r 0.0001 EZ1 1.43 EZ2 1.59 EZ3 2.06 EZ4 2.03 EZ12 1.12nn EZ13 1.01nn EZ14 1.45n EZ15 1.7 EZ1 þ EZ3 2.07 EZ12 þ EZ13 2.6 EZ12 þ EZ14 3.04n EZ13 þ EZ14 2.98n EZ12 þ EZ13 þ EZ14 3.17nn Control 2.16 SEM 0.22 P treatment r 0.0001
Maize stover Lucerne Maize stover Lucerne Lucerne Maize stover
0.2 0 0 0.1 0 0.1 0.1 0.3 0 0.3 0 0.1 0.2 0.3 0.1 0.6 295 621 87nn 281n 691n 390 421 546 141nn 287nn 171n 250nn 139nn 451 73 r 0.0001 60.4 38.6 66.8nn 51.5 35.6 43 55.8 41.3 72.0nn 57.5 55.6 45.8 66.3nn 41.9 5.5 0.003
2.49 2.18 1.73 1.69 1.66 1.55 1.58 1.42 2.92n 4.91nn 1.68 1.57 1.55 1.37 0.5 0.006
4.1 5.2 4.2nn 3.8nn 5.7 5.1 4.6n 5.1 3.7nn 4.3nn 4.8n 4 4.8 5 0.4 0.01
17 6 8 10 19 22 22 15 15 36n 5 8 5 11 5 0.01
n
Lucerne Maize stover
generally decreased ammonia concentration compared with control, most pronounced for EZ1þEZ3 (P¼0.0004). Glucose concentration increased for several treatments and most pronounced for EZ12þEZ13 and EZ12þEZ13þEZ14 (Pr0.0001). Lactic acid concentrations increased with several enzyme treatments compared with control, especially EZ1þEZ3 increased lactic acid concentration. Most enzyme treatments decreased pH significantly compared with control (Pr0.0001), especially the EZ1þ EZ3 treatment.
nn
Lucerne Maize stover Lucerne
n nn
Maize stover Maize stover Lucerne
55
3.2. Experiment 2
n
L-Lactic acid (g/kg of DM) Acetic acid (g/kg of DM) Propionic acid (mg/kg of DM) Butyric acid (mg/kg of DM) Glucose (g/kg of DM) Enzyme
Table 4 Fermentation products of maize stover and lucerne treated with enzymes (N ¼ 2).
NH3 (g/kg of DM)
pH
M.R. Dehghani et al. / Livestock Science 150 (2012) 51–58
Effect of enzyme treatments during ensiling on NDF digestion kinetics of silages is presented in Fig. 1, and enzyme treatments reduced NDF residues, but only had slight although in some cases significant effect on the residues after 96 h incubation. The potential degradability (Table 5) of NDF ranged from 0.20 to 0.73. Potential degradability decreased significantly for EZ1 and EZ1þ EZ3 treatments for maize stover (Claxxon) and EZ1þ EZ3 for maize stover (Bull). The potential degradability decreased for all enzyme treatments in grass clover compared with control. For lucerne silage the potential degradability of NDF showed the highest variation among treatments (from 0.56 to 0.73). Rate of degradation increased considerably for EZ3 and EZ1þEZ3 compared with control for maize stover (Bull) and resulted in increased effective degradability of NDF. For most other enzyme treatments effective degradability decreased compared with control. Based on the NDF concentrations in the silages and the effective degradabilities found, effective undegradable NDF was calculated as NDF (1 effective degradability of NDF) (Table 5). Effectively undegradable NDF was reduced by most enzyme treatments, and especially the combined EZ1 þEZ3 treatment reduced effectively undegradable NDF except for maize stover Claxxon. 4. Discussion 4.1. Experiment 1 Combination treatment with mixes of enzymes mostly resulted in larger effects compared with individual enzyme components, which might be due to synergistic effects or to the higher combined enzyme dose. Some enzyme treatments increased lactic acid concentration and decreased pH, and most pronounced for EZ12 for maize stover. The main activity of EZ12 was xylanase activity (Table 2). The combination of EZ12 þEZ14 was the most effective for increasing glucose and decreasing NDF concentration in maize stover. The combination of EZ1 and EZ3 (EZ1þ EZ3) with glucanase, b-glucanase and pectinase activity was most efficient in increasing fermentation products and in decreasing NDF concentration in lucerne silage. This combination of enzymes increased glucose and lactic acid and decreased pH and NDF and had the most pronounced effect compared with the other enzyme treatments. This is in accordance with Tengerdy et al. (1991) who found a cocktail of cellulase, hemicellulase and pectinase most enhancive in ensiling lucerne.
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M.R. Dehghani et al. / Livestock Science 150 (2012) 51–58
Table 5 NDF concentration and degradation kinetics of maize stover, grass clover and lucerne treated with selected enzymes (N ¼3). Potential degradability
ca (/h)
Feed
Treatment
NDF concentration (% DM)
Maize stover (Claxxon)
EZ1 EZ3 EZ1 þEZ3 Control
63.9 65.0 63.5 68.3
0.33n 0.38 0.32nn 0.43 0.02
0.028 0.029 0.027 0.028 0.004
0.19nn 0.23 0.17nn 0.25 0.007
51.7 50.3 52.8 51.4 0.5
EZ1 EZ3 EZ1 þEZ3 Control
52.1 56.2 52.0 56.0
0.45 0.44 0.40 0.48 0.03
0.0220 0.041n 0.035n 0.016 0.005
0.22 0.28n 0.25n 0.22 0.01
40.6nn 40.3nn 38.9nn 43.9 0.6
EZ1 EZ3 EZ1 þEZ3 Control
36.7 35.2 30.8 42.2
0.35nn 0.33nn 0.20nn 0.44 0.01
0.038 0.017n 0.042 0.048 0.004
0.23nn 0.15nn 0.13nn 0.31 0.01
28.4 29.9 26.8nn 29.2 0.4
EZ1 EZ3 EZ1 þEZ3 Control
18.5 18.7 15.3 25.0
0.65 0.73 0.56n 0.70 0.03
0.025 0.025 0.027 0.027 0.003
0.36n 0.40 0.30nn 0.40 0.01
11.8nn 11.2nn 10.7nn 15.0 0.2
SEM Maize stover (Bull)
SEM Grass clover
SEM Lucerne
SEM P
Feed Treatment Feed Treatment
r 0.0001 r 0.0001 0.03
0.2 0.5 0.04
Effective degradabilityb
r 0.0001 r 0.0001 r 0.0001
Effective undegradable NDFc (% DM)
r 0.0001 r 0.0001 r 0.0001
n Within a column and feed, means followed by star differ from control, nPo 0.05; nnP o0.01. SEM: standard error of Ls means. a Fractional rate of degradation. b Effective degradability calculated using 0.02/h passage rate. c Effective undegradable NDF calculated as NDF (% DM) (1 effective degradability).
Enzyme treatment of maize stover for silage decreased DM concentration compared with control probably due to a loss of dry matter during fermentation process and a higher production of volatile fermentation products, which evaporate during the drying process. Several of the fibrolytic enzyme treatments were effective in degrading cell wall carbohydrates (NDF) in the tested forages and therefore reduced NDF in the treated silages. NDF concentration generally decreased compared with control due to enzyme treatment for maize stover and lucerne, indicating that enzyme treatments were effective in solubilising NDF. Similar effects were found by Higginbotham et al. (1994), Shepherd and Kung (1996), Shepherd et al. (1995) and Spoelstra et al. (1992), however Mandebvu et al. (1999) and Kozelov et al. (2008) found that fibrolytic enzyme had no effect on cell wall concentration of bermudagrass and lucerne silage, respectively. This contradiction may be due to forage and enzyme type and level in these experiments. Despite the effect on NDF concentration, the digestibility of organic matter (in vitro) was not affected by enzyme treatment for lucerne and even decreased for some enzyme treatments for maize stover. That OM digestibility did not increase despite a reduction in NDF concentration which was probably caused by a solubilisation of only the easily digestible NDF part, as discussed below, and therefore OM digestibility was unaffected. That OM digestibility even decreased for some enzyme treatments of maize stover might be due to loss of fermentation products during the drying process. Lack of increase in OM digestibility despite
considerable reductions in NDF concentration when using enzymes as additive at ensiling is in accordance with the results of Nadeau et al. (2000a) and Van Vuuren et al. (1989). Fibrolytic enzymes generally increased L-lactic acid concentration in silage compared with control. This is probably due to a release of soluble and fermentable carbohydrates by fibrolytic enzyme degradation of NDF, and this supply of substrate increased the L-lactic acid production by the lactic acid bacteria. This is in accordance with Colombatto et al. (2004) who found that lactic acid concentration increased by enzyme treatment in corn silage, whereas Mandebvu et al. (1999) and Shepherd and Kung (1996) found no effect of fibrolytic enzymes on lactic acid concentration in bermudagrass silage and corn silage, respectively. The L-lactic acid concentrations found for some lucerne treatments were high, probably due to an abundant supply of substrate from the pectin degrading enzymes, combined with low DM concentrations which has allowed for an extended fermentation. Large variations in lactic acid and pH in lucerne silage were imposed by the enzyme treatments, and the lowest pH found for the enzyme treatment including pectinase was 0.8 pH units lower than the untreated control. Reduced pH in silage treated with enzymes is in accordance with results from Colombatto et al. (2004), Nadeau et al. (2000b), Stokes (1992) and Zahiroddini et al. (2004) who observed decreased pH in silage of corn, orchardgrass, mix of grass–legume, and barley, respectively, when fibrolytic enzymes were used for ensiling.
M.R. Dehghani et al. / Livestock Science 150 (2012) 51–58
Concentration of acetic acid was lower in several enzyme treated lucerne silages than in control. Acetic/ lactic acid ratio decreased in enzyme treated lucerne silage compared with control; whereby treatment with fibrolytic enzymes seems to result in a more homofermentative ensiling process. Nadeau et al. (2000b) reported more heterofermentic ensiling for lucerne silage compared to orchardgrass due to higher buffering capacity, but also that enzyme induced reduction in acetic acid concentration was more pronounced for lucerne than for ochardgrass consistent to this study. However, in maize stover silage, enzyme treatment increased acetic acid concentration, most pronounced for EZ12þEZ13 combining glucanase and xylanase, as xylose mainly is fermented to acetic acid (Fred et al., 1919). Several of the enzyme tested showed an improved fermentation resulting in decreased butyric acid concentration in both maize stover and lucerne silage compared with control. Enzyme treatment might increase glucose concentration as a result of the NDF degradation. Although the released glucose was the driver for the enhanced fermentation to especially lactic acid, not all released glucose was fermented and glucose concentration in the silage increased for some treatments. Shepherd et al. (1995) and Shepherd and Kung (1996) also reported that glucose increased in lucerne silage and corn silage, respectively, due to treatment with fibrolytic enzymes. The low pH in some of the treated silage seemed to prevent proteolysis and decrease NH3 concentration. Enzyme induced enhanced fermentation is beneficial in reducing protein degradation during ensiling and is in accordance with Zahiroddini et al. (2004) for barley silage and Nadeau et al. (2000a) for ochardgrass and lucerne silage. 4.2. Experiment 2 Treatment with fibrolytic enzyme could degrade the most easily digestible parts of NDF during ensiling and thereby reduce the NDF concentration in silage DM. NDF concentration was considerably lower in enzyme treated silages compared with control for the four silages selected for NDF degradation studies. Enzyme treatment decreased the potential degradability of silages compared with control, which is in accordance to the findings by Moharrery et al. (2009) that enzymes degrade the easiest degradable NDF whereas the concentration of indigestible NDF seems to keep constant in DM but increase as a proportion of NDF. When the digestible fraction of NDF decreased due to the effect of enzymes on cell wall carbohydrates, the indigestible NDF in total NDF increased (Fig. 1). This is in contrast to Stokes (1992) and Shepherd and Kung (1996) who found that enzyme treatment at ensiling increased in vitro DM digestion and had no effect on in vitro NDF digestion of grass–legume mix and corn silage, respectively, however in accordance with results from Nadeau et al. (1996) who also found that enzyme addition at ensiling especially solubilised the easily digestible fraction of NDF. The amount of iNDF to DM was approximately constant in the present study and total degradability of NDF decreased. Although effective
57
NDF degradability decreased except for maize stover (Bull) treatment EZ3 and EZ1 þEZ, the decreased NDF concentration can result in higher availability of nutrients in an in vivo situation. Effective undegradable NDF in per cent of DM was generally reduced by enzyme treatments, indicating that although no clear reduction was seen in indigestible NDF, effective undegradable NDF can be reduced and thereby an effective enzyme treatment can be expected to increase the concentration of cell soluble substances and reduce the amount of NDF and thereby increase DM digestibility in high producing dairy cattle with short rumen retention time. 5. Conclusion Addition of fibrolytic enzymes before ensiling increased lactic acid and glucose and decreased ammonia and butyric acid concentrations and decreased pH in silages. Data from this study further suggest that adding fibrolytic enzymes to forage at ensiling can solubilise some of the easy digestible parts of cell wall carbohydrates and thereby supply substrates for lactate fermentation. Therefore enzyme addition could be efficient to improve silage quality in forages which are difficult to ensile due to low sugar concentration and/or high protein concentration and buffer capacity. Enzyme treatment at ensiling with the enzymes used in the present study did not increase in vitro organic matter digestibility, but due to the reduced concentration of NDF, DM digestibility can be expected to increase in high producing dairy cattle with short rumen retention time. Conflicts of interest statement No conflicts of interest exits.
Acknowledgements We thank Mr. Torkild Jakobsen, Mrs. Inger Østergaard and Mrs. Anne Krustrup for help with the experimental work. The project was partly funded through the FFU (DANIDA) project ‘Income generation through market access and improved feed utilisation-production of quality beef and goat meat (IGMAFU-meat)’. Novozymes is acknowledged for supply of enzymes. References Colombatto, D., Mould, F.L., Bhat, M.K., Phipps, R.H., Owen, E., 2004. In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage. I. Effects of ensiling temperature, enzyme source and addition level. Anim. Feed Sci. Technol. 111, 111–128. Eun, J.S., Beauchemin, K.A., 2007. Assessment of the efficacy of varying experimental exogenous fibrolytic enzymes using in vitro fermentation characteristics. Anim. Feed Sci. Technol. 132, 298–315. Fahey Jr., G.C., Titgemeyer, E.C., Atwell, D.G., 1993. Postharvest treatment of fibrous feedstuffs to improve their nutritive value. In: Jung, H.G., Buxton, D.R., Hatfield, R.D., Ralph, J. (Eds.), Forage Cell Wall Structure and Digestibility, ASA, CSSA, SSSA, Madison, WI, pp. 715–766. Fred, E.B., Peterson, W.H., Davenport, A., 1919. Acid fermentation of xylose. J. Biol. Chem. 39, 347–384. Hansen, B., 1989. Determination of nitrogen as elementary N, an alternative to Kjeldahl. Acta Agric. Scand. 39, 113–118.
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Higginbotham, G.E., Mueller, S.C., Collar, C.A., Schultz, T.A., 1994. Effects of an enzyme addition on corn silage fermentation. Prof. Anim. Sci. 10, 163–168. Hvelplund, T., Weisbjerg, M.R., Hindrichsen, I.K., Madsen, J. The effect of adding exogenous enzymes at ensiling on nutrient availability in different forages. In: Proceedings of the 33th Annual Scientific Conference. Society of Animal Production, Tanzania. September 2009, in press. Kozelov, L.K., Lliev, F., Hristov, A.N., Zaman, S., McAllister, T.A., 2008. Effect of fibrolytic enzyme and an inoculant on in vitro degradability and gas production of low dry matter alfalfa silage. J. Sci. Food Agric. 88, 2568–2575. Kristensen, N.B., Danfær, A., Tetens, V., Agergaard, N., 1996. Portal recovery of intraruminally infused short-chain fatty acids in sheep. Acta Agric. Scand. Anim. Sci. 46, 26–38. Kung Jr., L., Stokes, M.R., Lin, C.J., 2003. Silage additives. In: Buxton, D.R., Muck, R.E., Harrison, J.H. (Eds.), Silage Science and Technology. Agronomy Monograph, 42. , ASA, CSSA, and SSSA, Madison, WI, pp. 305–360. Mandebvu, P., West, J.W., Froetschel, M.A., Hatfield, R.D., Gates, R.N., Hill, G.M., 1999. Effect of enzyme or microbial treatments of bermudagrass forages before ensiling on cell wall composition, end products of silage fermentation and in situ digestion kinetics. Anim. Feed Sci. Technol. 77, 317–329. McDonald, I., 1981. A revised method for the estimation of protein degradability in the rumen. J. Agric. Sci.(Cambridge) 96, 251–252. McDonald, P., Henderson, A.R., Heron, S.J.E., 1991. The Biochemistry of Silage, 2nd ed. Chalcombe Publications, Marlow, Bucks, UK. Mertens, D.R., 2002. Gravimetric determination of amylase-treated neutral detergent fiber in feeds with refluxing in beakers or crucibles: collaborative study. J. AOAC Intern. 85, 1217–1240. Moharrery, A., Hvelplund, T., Weisbjerg, M.R., 2009. Effect of forage type, harvesting time and exogenouse enzyme application on degradation characteristics measured using in vitro technique. Anim. Feed Sci. Technol. 153, 178–192. Nadeau, E.M.G., Buxton, D.R., Lindgren, E., Lingvall, P., 1996. Kinetics of cell-wall digestion of ochardgrass and alfalfa silage treated with cellulase and formic acid. J. Dairy Sci. 79, 2207–2216.
Nadeau, E.M., Buxton, D.R., Russell, J.R., Allison, M.J., Young, J.W., 2000a. Enzyme, bacterial inoculant, and formic acid effects on silage composition of orchardgrass and alfalfa. J. Dairy Sci. 83, 1487–1502. Nadeau, E.M., Russell, J.R., Buxton, D.R., 2000b. Intake, digestibility, and composition of orchardgrass and alfalfa silages treated with cellulase, inoculant and formic acid fed to lambs. J. Anim. Sci. 78, 2980–2989. SAS, 2003. In: User’s Guide: Statistics, Version 9.1edition SAS Institute, Inc., Cary, NC, USA. Shepherd, A.C., Kung Jr., L., 1996. Effects of an enzyme additive on composition of corn silage ensiled at various stages of maturity. J. Dairy Sci. 79, 1767–1773. Shepherd, A.C., Maslanka, M., Quinn, D., Kung Jr., L., 1995. Additives containing bacteria and enzymes for alfalfa silage. J. Dairy Sci. 78, 565–572. Spoelstra, S.F., Van Wikselaar, P.G., Harder, B., 1992. The effects of ensiling whole crop maize with a multi-enzyme preparation on the chemical composition of the resultant silages. J. Sci. Food Agric. 60, 223–228. Stokes, M.R., 1992. Effects of an enzyme mixture, an inoculant, and their interaction on silage fermentation and dairy production. J. Dairy Sci. 75, 764–773. Tengerdy, R.P., Weinberg, Z.G., Szakacs, G., WU, M., Linfen, J.C., Henk, L.L., Johnson, D.E., 1991. Ensiling alfalfa with additives of lactic acid bacteria and enzymes. J. Sci. Food Agric. 55, 215–228. Tilley, J.M.A., Terry, R.A., 1963. A two-stage technique for the digestion of forage crops. J. Br. Grassland Soc. 18, 104–111. Van Vuuren, A.M., Bergsma, K., Frol-Kramer, F., van Beers, J.A.C., 1989. Effects of addition of cell wall degrading enzymes on the chemical composition and the in sacco degradation of grass silage. Grass Forage Sci. 44, 223–230. Weisbjerg, M.R., Hvelplund, T., Søegaard, K., 2004. Prediction of NDF digestibility based on assumptions about true digestibility and endogenous loss of NDS. J. Anim. Feed Sci. 13 (Suppl. 1), 243–246. Zahiroddini, H., Baah, J., Absalom, W., McAllister, T.A., 2004. Effect of an inoculant and hydrolytic enzymes on fermentation and nutritive value of whole crop barley silage. Anim. Feed Sci. Technol. 117, 317–330.