Effects of an Enzyme Additive on Composition of Corn Silage Ensiled at Various Stages of Maturity' A. C. SHEPERD2 and L. KUNG, J R 3 Delaware Agricultural Experiment Station, Department of Animal Science and Agricultural Biochemistry, University of Delaware, Newark 19717
ABSTRACT
for use by the lactic acid bacteria during the fermentation process, resulting in silage with a lower final Corn forage at the milk, soft dough, and black layer pH and a higher lactic acid content. Enzyme additives stages of maturity ( 2 2 , 28, and 44% DM, respec- may also degrade portions of the plant cell wall durtively) was treated with an enzyme additive a t 1, 10, ing storage, thereby reducing fiber content and potenor 100 times the recommended dose and ensiled in tially improving animal performance. Although others laboratory silos at 26°C. By in vitro assay, the addi- have reported improvements in either fermentation tive conta:ined a full complement of cellulase and ( 3 , 15, 20) or animal production ( 2 3 ) when grass hemicellulase activities. "he pH and temperature op- silage was treated with enzyme additives, results of tima for cellulase and hemicellulase activities were experiments in which enzymes were added to corn 4.8 and 50"C, respectively. Regardless of dose, the silage have been conflicting ( 2 , 4, 21, 22, 2 3 ) . In additive had no effect on fermentation acids or previous studies, Cornzymem (Finnfeeds Internanitrogenous compounds in silage at any maturity; tional, Ltd., Schaumburg, IL), a commercial product however, high doses increased the glucose content of containing cellulase and hemicellulase activity from silage at the milk stage of maturity and increased Trichoderma reesei, had little effect on production of ethanol content at the soft dough stage. Across matu- silage acids, inconsistent effects on ADF and NDF rities, addition of the enzyme additive resulted in a content, and no effect on milk production when linear decrease in ADF, NDF, and hemicellulose con- treated silage was fed to lactating dairy cows ( 4 , 2 1). tent of corn silage but decreased the acid detergent Additionally, fiber digestion was unaffected by Cornlignin content of silage only a t the milk and black zyme@when measured in situ ( 4 ) or in vivo ( 2 1). layer stages of maturity. The enzyme additive had no This study was conducted 1) to evaluate the enconsistent effect on in vitro NDF digestion. zyme activities of Cornzymea, 2 ) to determine how ( Key words: corn silage, enzyme, maturity) stage of plant maturity affects the ability of Cornzyme@ to alter fermentation characteristics and fiber Abbreviation key: ADL = acid detergent lignin, BL content of corn silage, and 3 ) to determine whether = black layer stage of maturity, MK = milk stage of Cornzymem alters in vitro digestibility of corn silage. maturity, SDO = soft dough stage of maturity, x = times the recommended dose of Cornzymea (used with 1, 10, and 100). MATERIALS AND METHODS INTRODUCTlON
Enzymes that degrade plant cell walls have been added to silage to improve fermentation characteristics and animal performance. When soluble sugars are limiting, enzyme additives should improve fermentation characteristics by releasing soluble sugars
Received November 14, 1994. Accepted April 23, 1996. 'Published as Paper Number 1548 of the Delaware Agncultural Experiment !Station. Current address: Department of Dairy Science, University of Wisconsin, 1675 Observatory Drive, Madison 53706. 3Corresponding author. 1996 J Dairy Sci 79:1767-1773
Cornzymea was diluted in water, and enzyme activity was characterized as outlined by Wood and Bhat ( 2 7). Briefly, total cellulase activity was quantified by incubating the enzyme with a 1- x 6-cm strip of Whatman 1 filter paper (Whatman Corp., Clifton, N J ) in 0.05 mM citrate ( p H 4 . 8 ) for 1 h at 50°C. Carboxymethylcellulase activity was determined by incubation of the enzyme with carboxymethylcellulose ( 5 g / L in 50 mM sodium acetate, pH 4.8) for 30 min at 50°C. Cellobiohydrolase activity was determined by incubating the enzyme in a 1%(wt/vol) solution of cellulose (Sigma S3504; Sigma Chemical Co., St. Louis, MO) in 0.1 M sodium acetate ( p H 4 . 8 ) for 2 h at 50°C. In all assays, the dinitrosalicylic acid method was used to estimate the release of reducing sugars
1767
1768
SHEPERD AND KUNG
( 2 7 ) . Glucose was used as a standard, and the release of glucose equivalents was determined. To determine cellobiase activity, Cornzyme@ was incubated with 15 mM cellobiose in 0.05 M citrate buffer (pH 4.8) at 5OoC for 30 min. A glucose oxidase assay (kit 510; Sigma Chemical Co.) was used to determine glucose release. Endoxylanase activity was determined as described by Spoelstra et al. ( 2 2 ) . Cornzyme@was incubated with oat spelt xylan (X0627; Sigma Chemical Co.) in solution (30 g/L in 100 mM sodium acetate, pH 3.5) for 1 h at 39°C. Xylose standards were prepared, and the release of xylose equivalents was determined (2 8 ) . In order to evaluate the effect of pH on cellulase and xylanase activities, standard assays ( 2 7 1 were modified. Total cellulase and endoxylanase activities were quantified as previously described except that buffer pH was altered by adding either NaOH or HCl, resulting in final pH values of 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. Release of glucose or xylose equivalents were determined as previously described. To evaluate the effects of temperature on enzyme activity, pH of the assays were held constant at the previously determined optimum, and temperature was varied. Solutions of buffer and substrate were equilibrated at temperatures ranging from 4 t o 50°C for 15 min before addition of the enzyme solution and incubation for 1 h at the experimental temperature. Stoppers on tubes prevented evaporation of buffer. Release of reducing sugars was determined as previously described. Corn forage was harvested at the milk ( MK), soft dough ( SDO), and black layer ( BL) stages of maturity. At each harvest date, 50 plants from the same area within one field were cut manually and chopped in a forage harvester. Cornzyme@application rates of 1, 10 or 100 times ( x ) the dose recommended by the manufacturer were 0.22, 2.2, and 22 Wtonne of wet forage, respectively. Cornzyme@was diluted in distilled water, and 1 L of water (untreated) or water and enzyme solution was sprayed onto 40 kg of
forage. Forage was mixed by hand during treatment, and approximately 500 g were packed into 5- x 30-cm polyvinyl chloride laboratory-scale silos (four silos per treatment). Silos were stored for 60 d at 26°C. On each sampling day, silos were opened, and the contents were mixed thoroughly. A representative 25 g of forage or silage were homogenized with 225 g of deionized water for 1 min and filtered through Whatman 54 filter paper. A pH was determined on the homogenate, and a portion of the water extract was acidified with 6N HCl and frozen for later analysis. Acetic acid was determined by gas chromatography (model 5890; Hewlett Packard, Avondale, PA) and 530 gm of Carbowax@ M (Supelco, Inc., Bellefonte, PA). The chromatograph oven was programmed as follows: 70°C for 1 min, then a 5°C increase/min t o lOO"C, a 45°C increasdmin to 170"C, and a final holding time of 5 min. A phenolhypochlorite assay was used t o determine ammonia N content (17), and glucose was determined using a glucose oxidase assay (kit 5 10; Sigma Chemical Co. ). L-Lactic acid and D-lactic acid were analyzed by an enzymatic method (kit 826-UV; Sigma Chemical Co.). For analysis of D-lactic acid, L-lactic acid dehydrogenase was replaced with a similar amount of D-lactic acid dehydrogenase (L-9636; Sigma Chemical Co.). L-Lactic acid (L-2250; Sigma Chemical Co.) and D-lactic acid (L-1000; Sigma Chemical Co.) were used for standards for their respective assays. Total lactic acid concentration was the sum of the concentrations of both isomers of lactic acid. Forage and silage DM were determined by air drying at 55°C for 48 h in a forced-air oven. Samples were then ground through a 1-mm screen prior to nonsequential analysis for ADF ( 7 ) and NDF ( 2 5 ) . Crude protein content was calculated ( N x 6.25) after determination of N ( 1).The acid detergent lignin ( ADL) procedure was used t o estimate the lignin content of forages ( 7 ) . Hemicellulose content was calculated as the difference between NDF and ADF. Extent of in vitro NDF digestion at 48 h was determined as described by
TABLE 1. Fibrolytic activity of
[email protected] Enzyme
Substrate
Measured product
Specific activity2
&Glucosidase (EC 3.2.1.21) Endo-l,4-@-glucanase(EC 3.2.1.4) Cellobiohydrolase (EC 3.2.1.91) Endoxylanase (EC 3.2.1.8)
Cellobiose CMC3 Cellulose Oat epelt xylan
Glucose equivalents Glucose equivalents Glucose equivalents Xylose equivalents
3.4 8.9 1.3 44.6
1Finnfeeds International, Ltd. (Schaumburg, IL). 2Millimoles per minute per gram of protein.
carboxymethylcellulose. Journal of Dairy Science Vol. 79, No. 10, 1996
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CORN PLANT MATURITY AND AN ENZYME ADDITIVE
Goering arid Van Soest ( 7 1. Ruminal fluid was collected approximately 4 h after feeding from a fistulated steer fed a diet consisting of alfalfa hay and corn silage (50:50). Statistical Analysis
Silage dlata were analyzed as a factorial experiment using the general linear models procedure of SAS (18).Stage of maturity and enzyme dose were tested for linear and quadratic effects. When a significant F test was detected, means were compared by Tukey's test. Extent of in vitro digestion was analyzed at each time point by ANOVA using the general linear models procedure of SAS ( 18). When a significant F test was detected, means were compared by Tukey's test. Significance was declared at P < 0.05 unless otherwise noted. RESULTS
Cornzynne@contained 1.39 x g of proteidml. Theoretical application rates resulted in additions of 0.31, 3.06, and 30.60 g of proteidtonne of wet forage at the lx, lox, and lOOx doses, respectively. Cornzyme@ contains the three required enzymes [endoglucanase, exoglucanase (cellobiohydrolase), and 0-glucosidase] that act synergistically to hydrolyze cellulose into its monomer unit, glucose ( 13, 2 8) (Table 1). Of the three enzymes, endoglucanase activity was greatest, followed by lower activity of 0glucosidase and cellobiohydrolase. Cornzyme@ also contains substantial hemicellulase (xylanase) activity. Others ( 1 0 ) have reported that enzyme systems from T. reesei often contain hemicellulases. Similar to our findings, Khan et al. ( 1 0 ) reported about 10 times greater xylanase than cellulase activity in a n enzyme extract from T. reesei. In addition to xylanase activity, Khan et al. ( 1 0 ) reported that a T. reesei enzyme complex also contained acetyl-xylan
esterase, a-glucuronidase, 0-D xylosidase, a-Darabinose, and 0-D glucosidase activity. Spoelstra et al. ( 2 2 1 reported that some commercial (hemi-)cellulase preparations may contain amylolytic activity; however, Selmer-Olsen ( 19 ) conducted studies with enzyme additives from T. reesei and Aspergillus spp. that contained no amylolytic activity. Although we did not determine amylolytic activity, Cornzyme@ contained negligible amounts of amylolytic activity (R. Treacher, Finnfeeds, International, Ltd., 1995, personal communication). Results from our in vitro assay were in agreement with other data (11, 28) reporting that the cellulase and hemicellulase activities from T. reesei were maximal at a pH of approximately 4.8 and a temperature of approximately 50°C. Thus, these data have been excluded from this report. However, temperature and pH optima for these enzymes may play a crucial role in their effectiveness as silage additives because the pH of corn silage can decrease to less than 4 within 48 h of ensiling, and seldom do silages maintain prolonged temperatures of 50°C. The optimal pH for cellulase activity is important because adsorption of the enzyme to substrate and hydrolysis of the substrate appear to be strongly affected by pH ( 11). In our experiments, more than 70% of enzyme activity remained between a pH of 3.5and 4.0 ( a t 50"C), but, a t 26"C, enzyme activity was decreased by more than 70%. At temperatures below 1O"C, enzyme activity was less than 10% of that at 50°C. For most forages, advanced maturity increased DM and fiber content and DM yield, but protein content and digestibility tended to decrease; however, fiber content of whole-plant corn, tended to decrease with maturity because of the increase in grain content ( 6 1. Flachowsky et al. ( 5 ) reported that a n ear of corn contained only 29% of the total plant DM when corn was harvested at the early MK but contained about 59% of the total DM when the plant was harvested a t the hard dough stage of maturity. Thus, the fiber
TABLE 2. Composition of fresh corn forage prior to ensiling but after treatment with water or Cornzyme".' Maturity
DM
NDF
(a) SE Milk Soft dough Black layer
22.0 0.3 28.0 1.0 44.0 1.0
fi
ADL2
ADF
SE
51.1 1.3 43.2 2.7 42.6 2.9
E
Hemicellulose3 CP (5% of
SE
31.7 1.8 24.5 1.8 22.8 1.9
fi
SE
3.67 0.25 2.30 0.14 2.47 0.33
DM) X
SE
19.4 1.3 18.9 1.4 19.7 1.4
X
Glucose
SE
8.72 0.24 8.77 0.22 8.76 0.15
Ti
SE
4.90 0.39 4.22 0.44 4.47 0.50
1Finnfeeds International, LM. (Schaumburg, IL). 2Acid detergent lignin. 3NDF ADF.
-
Journal of Dairy Science Vol. 79, No. 10, 1996
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SHEPERD AND KUNG
content of the whole plant decreased even though the stem continued to mature. In our study, NDF, ADF, and ADL content of whole-plant corn forage tended t o decrease with maturity (Table 2). Fiber content decreased most between MK and SDO, but remained constant between SDO and BL. The CP and glucose content of forages remained fairly constant with increasing stages of maturity. Table 3 shows the DM and fermentation characteristics of corn silages after 60 d of ensiling. Stage of maturity had quadratic effects on DM, ammonia N, acetic acid, and total lactate concentrations but no effect on ethanol content of silages. Interactions between stage of maturity and enzyme dose were observed for silage pH and acetic acid, ethanol, and glucose content. Treatment with Cornzymem reduced the pH of MK silage by approximately 0.4units relative t o the untreated silage; however, pH did not differ among enzyme doses. In contrast, the higher doses of Cornzyme@had no effect on the pH of SDO or BL silages. Enzyme treatment had no effect on total lactate, acetic acid, ammonia N content of silages, regardless of maturity. Enzyme treatment had no effect on ethanol content of MK or BL silages, but, relative to untreated silage, the lox and lOOx doses
increased ethanol content by more than 200% in SDO silage. In contrast, an enzyme dose of lOOx increased glucose content only in MK silage. Enzyme additives that hydrolyze the fibrous components of the plant cell wall and release glucose can improve fermentation (8, 9, 20); however, corn normally contains large amounts of soluble sugars and ferments rapidly, making it an ideal crop for silage (14). In our study, a general lack of effect of enzyme on fermentation end products agreed with data of other researchers ( 2 , 41, who found no effect of CornzymeQ on silage fermentation when corn forage was harvested at mid to late stages of maturity. Cumulatively, these findings confirm the fact that fermentable substrate is usually not a limiting factor for corn silage fermentation. An increase in ethanol content caused by enzyme treatment has been reported by others (22,24).Spoelstra et al. (22) reported that substantial amylolytic activity from their hemicellulase preparation resulted in the degradation of starch and subsequent production of ethanol and an aerobically unstable silage. Substantial production of ethanol in silage would also be unfavorable because yeast produces large quantities of C O 2 during the production of ethanol, which would lead to losses in DM content.
TABLE 3. Fermentation characteristics of corn silage harvested at three stages of maturity and treated with Cornzyme@lat 0, 1, 10, and 100 times ( x) the dosage2 recommended by the manufacturer. Maturity and dosage
DM
PH
NH3 N
Total lactate
Acetic acid ( % of
(%)
Ethanol
Glucose
DM)
Milk 0
lx lox lOOx Soft dough 0
lx lox lOOx Black layer 0
lx lox lOOx Pooled SE3 Contrast4 Maturity ( M ) Enzyme ( E ) MxE
25.8 25.8 25.3 25.8
4.058 3.58bd 3.58bd 3,57bd
0.06 0.05 0.06 0.05
7.4 8.7 8.4 7.4
1.27 1.41 1.61 1.26
l.22hd 1.23be 1.12hd 1.60b
0.67h 0.62h 1.27b 2.608
31.9 31.3 30.0 29.9
3.42Cd 3.36d 3.45m 3.47m
0.04 0.05 0.05 0.05
6.1 7.9 7.1 6.9
0.86 1.04 0.92 0.98
0.45d 0.49d 2.708 1.55be
0.52h O.5lh 0.34h 0.17c
43.1 43.1 42.0 44.0 1.1
3.68b 3.69b 3.66bc 3.63bC 0.10
0.06 0.06 0.06 0.05 <0.01
4.9 4.6 5.3 4.7 0.9
0.83 0.70 0.80 0.84 0.16
0.74cd 0.95hd 1.38h 1.22kd 0.31
0.56h 0.66h 1.08be 0.96h 0.44
Q Q
NS
Q Q
<0.01
Q
NS NS
Q
NS NS
a,b,c.dMeans within a column and maturity with unlike superscripts differ ( P 'Finnfeeds International, Ltd. (Schaumburg, IL). %mount of Cornzymem relative to recommended dose of the manufacturer. 3n = 4. 4L = Linear; Q = quadratic response ( P c 0.05). N S = P z 0.10. Journal of Dairy Science Vol. 79, No. 10, 1996
0.05).
Q
NS
NS NS
co.01
Q
Q
L <0.01
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CORN PLANT MATURITY AND AN ENZYME ADDITIVE
Table 4 shows the CP and structural carbohydrate sistently improved silage quality. Sheperd and Kung components of untreated and treated silages. Increas- ( 2 1) reported that Cornzyme@decreased the fibrous ing the stage of maturity caused quadratic changes in components of corn silage in laboratory silos but not all components. As expected, enzyme treatment had in farm-scale bag silos. Spoelstra et al. ( 2 2 ) reported no effect on CP content of silages. Because plant cell that corn silage harvested at the dough stage of matuwalls change with maturity, there was reason to sus- rity and treated with cellulase and hemicellulase enpect that interactions might exist between the ability zymes had as much as 25% of the NDF degraded by of Cornzyme@ t o alter fiber content of silage and the enzymes. Stokes and Chen ( 2 4 ) reported that an forage maturity. For example, both NDF and ADF enzyme and bacterial additive reduced the fibrous contents of grass silage were reduced by enzyme components (cellulose and hemicellulose) of corn treatment, but the extent was greater for immature silage that had been harvested in mid-October (DM forage than for mature forage ( 2 6 ) . In contrast, en- content, -30%; NDF content, -54%). In contrast, zyme treatment did not alter the fiber content of a Chen et al. ( 4 ) reported no effect of enzyme addition barley and vetch silage mixture at various stages of on the fiber content of corn silage. maturity ( 1 2 ) . In the current study, no interactions Cornzyme@had no effect on the extent of in vitro were observed between enzyme dose and stage of NDF digestion of silage harvested at MK or BL maturity for any structural carbohydrate component (Table 5). When forage was harvested at SDO, only except ADL; however, the main effect of enzyme dose the l o x treatment increased NDF digestibility. For caused linear decreases in the NDF, ADF, and most grasses and legumes, the stem to leaf ratio hemicellulose contents of silages. Addition of cellulase increases as the plant matures, and, because the leaf or hemicellulase enzymes to corn silage has not con- is more readily digestible, the digestibility of the
TABLE 4. Crude protein and structural carbohydrate content of silage harvested at three stages of maturity and treated with CornzymeQ' at 0, 1, 10, and 100 times ( x) the dosage2 recommended by the manufacturer. Maturity and dose
CP
NDF
ADF
Hemicellulose3
ADL4
(5% of DM) Milk 50.0 46.3 47.4 42.9
30.0 28.6 29.7 27.7
20.1 17.7 17.7 15.3
4.88a 4.32ab 3.32bd 3.67bc
8.1 8.2
34.7 36.4 35.1 32.0
22.4 23.1 22.4 21.4
12.3 13.3 12.6 10.5
2.7lcde 3.44bcd 2.95Cde 3.Olcde
8.3 8.2 8.2 8.4 0.4
37.4 35.4 35.5 32.1 2.3
22.1 19.9 20.2 17.0 1.4
15.2 20.1 15.3 15.1 1.8
2.76cde 2.89cde 2.41de 2.03e 0.43
Q
Q
Q
Q
NS
L NS
8.0
0
lx lox lOOx Soft dough
8.3 8.3 8.1 7.8
0
lx lox lOOx Black layer 0
lx lox lOOx Pooled SE5 Contrast6 Maturity ( M ) Enzyme ( E ) M x E
8.0
NS NS
L
NS
L
Q Q
<0.01
a,b.c,d,eMeanswithin a column with unlike superscripts differ ( P < 0.05). 'Finnfeeds International, Ltd. (Schaumburg, IL). 2Amount of Cornzyme@relative to recommended dose of the manufacturer. 3NDF - ADF. 4Acid detergent lignin. 5n = 4. 6L = Linear; Q = quadratic response ( P < 0.05). NS = P > 0.10. Journal of Dairy Science Vol. 79, No. 10, 1996
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SHEPERD AND KUNG
TABLE 5. Extent of 48-h in vitro NDF digestion (percentage of NDF) of silage harvested a t various stages of maturity and treated with Cornzymeml a t 0,1,10, and 100 times ( x) dosage recommended by the manufacturer. Enzyme dose2 Maturity
0
lx
lox
lOOx
SE3
Milk SoR dough Black layer
57.2 58.1b 54.3
59.2 59.3ab 55.5
56.0 62.9a 55.2
57.3 55.7b 56.7
2.0 1.1 0.8
asbMeans within a column and maturity with unlike superscripts differ ( P < 0.05) 'Finnfeeds International, Ltd. (Schaumburg, IL). 2Amount of Cornzymem relative to recommended dose of the manufacturer. 3n = 4.
plant decreases ( 1 6 ) . Corn, however, differs from most grasses, and, although the digestibility of the stem decreases with maturity, grain content increases, thereby maintaining relatively constant digestibility as the plant matures ( 15). From our data and for the maturity stages studies, it was apparent that changes in fiber composition had minimal effects on NDF digestion. CONCLUSIONS
Cornzyme@contained a complex of cellulase enzymes and hemicellulase activity. When added t o corn silage at various stages of maturity, Cornzyme@had minimal effects on the fermentation of silage, except for increased ethanol in silage harvested at SDO and increased glucose in silage harvested at MK. Increasing doses of Cornzyme@ linearly decreased ADF, NDF, and hemicellulose content of silages across maturities but effects on in vitro NDF digestion were inconsistent. These findings suggest that Cornzyme@ could be used to decrease the fiber content of silages, but has minimal value relative to improving silage fermentation. ACKNOWLEDGMENTS
The authors thank Tony Timko and the farm crew at the University of Delaware for their assistance with forage planting and harvesting. We also thank C. M. Golt and A. M. Smagala for technical assistance and John Pesek for statistical assistance. REFERENCES 1Association of Official Analytical Chemists International. 1990. Official Methods of Analysis. 15th ed. AOAC, Arlington, VA. 2 Autrey, K M., T. A. McCaskey, and J. A. Little. 1975. Cellulose digestibility of fibrous materials treated with Trichoderma uiride cellulase. J. Dairy Sci. 58:67.
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3 Chamberlain, D. G., and S. Robertson. 1992. The effects of the addition of various enzyme mixtures on the fermentation of perennial ryegrass silage and on its nutritional value for milk production in dairy cows. Anim. Feed Sci. Technol. 37:257. 4 Chen, J . , M. R. Stokes, and C. R. Wallace. 1994. Effects of enzyme-inoculant ~ y s t e mon~ preservation and nutritive value of haycrop and corn silages. J. Dairy Sci. 77:501. 5 Flachowsky, G., W. Peyker, A. Schneider, and K. Henkel. 1993. Fibre analyses and in aacco degradability of plant fractions of two corn varieties harvested a t various times. Anim. Feed Sci. Technol. 43:41. 6Ganoa, K. H., and G . W. Roth. 1992. Kernel milk line as a harvest indicator for corn silage in Pennsylvania. J . Prod. Agric. 5:519. 7Goering, H. K., and P. J. Van Soest. 1970. Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). Agric. Handbook No. 379. ARS-USDA, Washington, DC. 8 Henderson, A. R., and P. McDonald. 1977. The effect of cellulase preparations on the chemical changes during the ensilage of grass in laboratory silos. J . Sci. Food Agric. 28:468. 9Henderson, A. R., P. McDonald, and D. Anderson. 1982. The effect of a cellulase preparation derived from Trschoderna uiride on the chemical changes during the ensilage of grass, lucerne and clover. J. Sci. Food Agric. 33:16. 10 Khan, A. W., K. A. Lamb, and K. G. Johnson. 1989. Formation of enzymes required for the hydrolysis of plant cell wall polysaccharides by Trichoderma reesei. Appl. Microbiol. Biotechnol. 5: 49. 11 Kim, D. W., J . H. Yang, and Y. K. Jeong. 1988. Adsorption of cellulase from Trichoderma uiride on microcrystalline cellulose. Appl. Microbiol. Biotechnol. 28:148. 12Kung, L., Jr., B. R. Carmean, and R. S. Tung. 1990. Microbial inoculation and cellulase enzyme treatment of barley-vetch silage harvested a t three maturities. J . Dairy Sci. 73:1304. 13 Marsden, W. L., and P. P. Gray. 1984. Enzymatic hydrolysis of cellulose in lignocellulosic materials. CRC Rev. Biotechnol. 3: 235. 14McDonald, P., A. R. Henderson, and S.J.E. Heron. 1991. The Biochemistry of Silage. 2nd ed. Chalcombe Publ., Bucksburn, England. 15 McHan, F. 1986. Cellulase-treated coastal bermudagrass silage and production of soluble carbohydrates, silage acids, and digestibility. J. Dairy Sci. 69:431. 16Nelson, C. J., and L. E. Moser. 1994. Plant factors affecting forage quality. Page 115 in Forage Quality, Evaluation, and Utilization. G. C. Fahey, Jr., M. Collins, D. R. Mertens, and L. E. Moser, ed. Am. Soc. Agron., Crop Sci. Soc. Am., and Soil Sci. Soc. Am., Madison, WI. 17 Okuda, H., S.Fuji, and Y. Kawashima. 1965. A direct colorimetric method for blood ammonia. Tokushima J. Exp. Med. 12:ll. 18 SAS@User's Guide: Statistics, Version 6 Edition. 1988. SAS Inst., Inc., Cary, NC.
CORN PLANT MATURITY AND AN ENZYME ADDITIVE
19 Selmer-Olsen, I. 1994. Enzymes a s silage additives for grassclover mixtures. Grass Forage Sci. 49:305. 20 Selmer-Olsen, I., A. R. Henderson, S. Robertson, and R. McGinn. 1993. Cell wall degrading enzymes for silage. 1. The fermentation of enzyme-treated ryegrass in laboratory silos. Grass Forage Sci. 48:45. 21 Sheperd, A. C., and L. Kung, J r . 1996. An enzyme additive for corn silage: effects on silage composition and animal performance. J. Dairy Sci. 79:1760. 22 Spoelstra, S. F., P. G. van Wikselar, and B. Harder. 1992. The effects of ensiling whole crop maize with a multi-enzyme preparation on the chemical composition of the resulting silages. J. Sci. Food Agric. 60:223. 23 Stokes, M. R. 1992. Effects of a n enzyme mixture, a n inoculant, and their interaction on silage fermentation and dairy production. J. Dairy Sci. 75:764.
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24Stokes, M. R., and J. Chen. 1994. Effects of a n enzymeinoculant mixture on the course of fermentation of corn silage. J. Dairy Sci. 77:3401. 25Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583. 26Van Vuuren, A. M., K. Mergsma, F. Frol-Kramer, and J.A.C. van Beers. 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. 27Wood, T. M., and K. M. Bhat. 1988. Methods for measuring cellulase activities. Methods Enzymol. 160237. 28 Woodward, J . 1991. Synergism in cellulase systems. Bioresources Technol. 36:67.
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