TY - JOUR
AU - Beauchemin, K. A.
AB - ABSTRACT With the objective of developing a rational approach for the selection of feed enzymes for ruminants, 22 commercial enzyme products were examined in terms of protein concentration, enzymic activities on model substrates, and hydrolytic capacity, the latter determined from the release of reducing sugars from alfalfa hay and corn silage. An in vitro ruminal degradation assessment was carried out using the same substrates, untreated or treated with the 22 enzyme products at 1.5 μL/g forage DM. Stepwise regressions were then performed to establish relationships between these factors. Protein concentration and enzymic activities explained at least 84% (P < 0.01) of the variation in the release of reducing sugars from alfalfa and corn silage. Alfalfa DM degradation after incubation with ruminal fluid for 18 h was positively related to xylanase activity (R2 = 0.29, P < 0.01), but the same activity was negatively related to DM degradation of corn silage (R2 = 0.19, P < 0.05). Protease activity explained a further 10% of the alfalfa DM degradation (P < 0.10). Following sequential steps involving the determination of rate and extent of DM and fiber degradation, the best candidates for alfalfa and corn silage were selected. Enzyme products effective with alfalfa hay seemed to exert part of their effect during the pretreatment period, whereas enzymes effective with corn silage worked exclusively after ruminal fluid was added. This finding suggests that different modes of action of exogenous enzymes are attacking different substrates and may partly explain enzyme-feed specificity. In alfalfa, it seems that effective enzymes work by removing structural barriers that retard the microbial colonization of digestible fractions, increasing the rate of degradation. In corn silage, effective enzymes seem to interact with ruminal enzymes to degrade the forage more rapidly, which is consistent with previous findings of synergism between exogenous and ruminal enzymes. Introduction The use of fibrolytic enzymes for ruminant diets has attracted interest, following trials in which positive responses in nutrient digestion and animal performance were observed (Beauchemin et al., 1995; Schingoethe et al., 1999). However, the mode of action of these enzymes in ruminants has not been clearly identified thus far, and the response to enzyme supplementation appears to depend on such factors as enzyme activities, diet, and animal physiological status. Enzyme products currently used in animal nutrition are mixtures of enzymes with differing characteristics (Vahjen and Simon, 1999). The biochemical properties of these enzymes may dictate the nature of the responses, but they are often overlooked or poorly defined before use (McAllister et al., 2001). It is yet unclear which, if any, enzyme activity limits the rate and extent of degradation in the rumen. Proper enzyme characterization should therefore be the first step in enzyme selection. However, enzyme activities are generally determined based on the release of hydrolysis products from model substrates, which may not resemble natural substrates. Hence, determination of the hydrolytic capacity of enzymes on natural substrates is also necessary. Finally, the enzyme products should be evaluated in the presence of ruminal fluid. Because of the impossibility of testing all enzymes in vivo, in vitro screening systems are suitable alternatives for identifying the most promising enzymes. With the objective of developing a rational approach for the selection of feed enzymes for ruminants, the present study examined 22 commercial enzyme products for their activity profiles, hydrolytic capacity, and in vitro ruminal degradation of forages commonly used in ruminant diets. Correlations were then performed to establish relationships between these factors. Two products were selected after sequential in vitro degradation steps, and their effects on rate and extent of in vitro forage degradation were determined. Material and Methods Enzyme Products The 22 enzyme mixtures used in this study were commercial products from various manufacturers supplied by Cargill, Inc. (St. Louis, MO). The products were named according to an experimental code (from RT1180 to RT1201). Also, three commercial products of known efficacy were included as positive controls. They were Promote N.E.T. (Lot MO-E100), Promote Dairy (PD, Lot 15524), and Promote Beef (PB, Lot 15525), all supplied by Cargill, Inc. Protein Concentration The amount of protein present in the commercial products was determined using the Bio-Rad DC protein determination kit (Bio-Rad Laboratories, Hercules, CA), with bovine serum albumin as the standard. Five microliters of suitably diluted enzymes was added to microtiter plates, followed by 25 μL of Bio-Rad reagent A and 200 μL of reagent B. The reaction was allowed to proceed for 15 min at room temperature, and absorbance was read at 630 nm using a MRX-HD plate reader (Dynatech Laboratories, Inc., Chantilly, VA). Enzymic Activities The enzyme products were analyzed for their main and side activities at 39°C and pH 6.0, to reflect ruminal conditions. Polysaccharidase activities were determined in triplicate using substrate solutions or suspensions (1% wt/vol) in distilled water. Xylan (from birchwood or from oat spelts), carboxymethylcellulose (medium viscosity), Sigmacell 50, lichenan, laminarin, and soluble starch (all obtained from Sigma Chemicals, St. Louis, MO) were used for determination of xylanase (EC 3.2.1.8), endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91), β-1,3-β-1,4-glucanase (EC 3.2.1.73), β-1,3-glucanase (EC 3.2.1.6), and α-amylase (3.2.1.1), respectively. In addition, barley β-glucan, xyloglucan (from tamarind seeds), and wheat arabinoxylan were obtained from Megazyme International Ltd. (Wicklow, Ireland). Suitably diluted enzyme (50 μL) and substrate solutions (450 μL) were incubated for 5 to 60 min, depending on the activity, and experimental protocols followed those outlined by Wood and Bhat (1988). The reaction was terminated by adding 2 vol of Somogyi-Nelson reagent (Somogyi, 1952) and boiling for 10 min. Reducing sugars were determined colorimetrically at 630 nm. One unit of activity was defined as the amount of enzyme required to release 1 μmol equivalent xylose or glucose per minute per gram of enzyme product, under the conditions of the assay. Glycosidase activities measured were β-D-glucosidase (EC 3.2.1.21), β-D-xylosidase (EC 3.2.1.37), α-L-arabinofuranosidase (EC 3.2.1.55), β-D-galactosidase (EC 3.2.1.23), and acetyl esterase (EC 3.1.1.6) using 1 mM solutions of p-nitrophenyl (obtained from Sigma) derivatives as described in Wood and Bhat (1988). One hundred microliters of each substrate was incubated (n = 6) with appropriately diluted enzyme (12.5 μL) and buffer (37.5 μL) at 39°C for 30 min, except for acetyl esterase activity. Upon incubation, the reaction was terminated by addition of 150 μL of 0.4 M glycine-NaOH buffer (pH 10.8) and the absorbance was measured at 420 nm. For acetyl esterase determination, sequential readings were taken at 0, 5, 10, and 15 min of incubation and activity was calculated based on the increase in absorbance at 340 nm. One unit of activity was defined as the amount of enzyme required to release 1 μmol of nitrophenol per minute per gram of enzyme product. Protease activity was determined using a radial diffusion assay method (Brown et al., 2001). Ten milliliters of a 1% (wt/vol) molter agar (Fermtech Agar, EM Science, Gibbstown, NJ), prepared in citrate-phosphate buffer (0.1 M, pH 6.0) and containing 0.5% (wt/vol) gelatin (Fisher Scientific, Fair Lawn, NJ) as substrate was poured on petri dishes (90-mm diameter). Sodium azide (0.01% wt/vol final concentration) was also included to prevent microbial growth. Upon agar solidification, a 6-mm well was made in each plate using a cork borer, and 5 μL of undiluted enzyme product plus 20 μL of distilled water was added. The plates were incubated at 39°C for 16 h. At the end of the incubation period, the unhydrolyzed gelatin was precipitated by addition of a saturated ammonium sulfate solution. The clear radial areas around the wells (denoting areas degraded by the enzymes) were measured by two independent observers using an electronic digital caliper (Traceable, Model No. 62379-531, Control Company, Friendswood, TX). The protease activity was then expressed in terms of millimeters of gelatin degraded, after correction for the well's diameter. Release of Reducing Sugars from Natural Substrates The hydrolytic potential of the enzyme products was determined in triplicate by measuring the reducing sugars released from 25 mg of alfalfa hay or corn silage (freeze-dried and milled to pass a 1-mm screen) after 15 min of incubation at 39°C and pH 6.0 (450 μL of 0.1 M citrate-phosphate buffer) with enzyme product (50 μL). Enzymes present in powder form were diluted 250-fold with distilled water (i.e., 0.1 g in 25 mL), whereas enzymes in liquid form were diluted 25-fold. This was based on preliminary experiments in which powdered enzymes were on average 10-fold more potent in releasing reducing sugars than their liquid counterparts. Before freeze-drying, the substrates were washed with distilled water for 2 h at room temperature to extract soluble components. Blanks containing only substrates were included for correction. The reducing sugars released were expressed in micrograms of glucose equivalents per milligram of enzyme product added. In Vitro Ruminal Degradation Assessment A series of experiments were carried out to select the most promising enzyme candidates. In Exp. 1, 1-g DM aliquots of alfalfa hay or corn silage (± 20 mg, dried and milled to pass a 2-mm screen) were weighed into 125-mL fermentation bottles (Wheaton Scientific, Millville, NJ). The same batches of alfalfa hay and corn silage were used throughout the series of experiments. The alfalfa hay used contained 382.0 and 252.4 g/kg DM of NDF and ADF, respectively, whereas the corn silage contained 467.4 and 254.1 g/kg DM of NDF and ADF, respectively. The 22 enzyme candidates were applied at a rate of 1.5 mg/g DM forage, 20 h before inoculation with ruminal fluid. Three other commercial products, Promote N.E.T. (Lot MO-E100), Promote Dairy (Lot S-15524), and Promote Beef (Lot S-15525), were included as positive controls. Exactly 125 mg of each enzyme product was dissolved in 50 mL of distilled water, and 0.6 mL was added to each bottle. Treatments were weighed in triplicate. Three hours later, 40 mL of anaerobic buffer medium, prepared as outlined by Goering and Van Soest (1970) and adjusted to pH 6.0 using 1 M trans-aconitic acid (Sigma Chemicals), was added, and bottles were stored at 25°C overnight. Ruminal fluid was obtained from three lactating dairy cows fed a corn silage-based total mixed ration. Feed was withdrawn from the feeders 4 h before the fluid was collected. Whole ruminal contents were strained through four layers of cheesecloth under a continuous stream of CO2, and the fluid was transported to the laboratory in prewarmed thermal flasks. Ten milliliters of ruminal fluid was inoculated in each bottle, already prewarmed to 39°C. Controls containing only substrate or only ruminal fluid were also included in triplicate. Bottles were incubated at 39°C for 18 h, and undegraded residues were immediately filtered through preweighed sintered glass crucibles (porosity 1, 100- to 160-μm pore size). Residues were dried at 110°C for 24 h to determine apparent dry matter degradation (DMD), expressed as grams per kilogram. The ranking of enzyme products on each feed was determined based on their relative increase in DMD with respect to the controls. Experiment 2. Based on the results obtained in Exp. 1, four enzyme products (RT1181, RT1183, RT1184, and RT1197) were chosen for further evaluation. Promote Dairy (Lot S-15524) was also included. The Daisy II in vitro fermentation system (ANKOM Corp., Fairport, NY) was used to examine the rate and extent of DM and fiber degradation of forages treated with these enzyme products. Five hundred milligrams (± 20 mg) of each substrate (alfalfa hay or corn silage) was weighed into artificial fiber bags (#F57, ANKOM Corp.), which were then heat-sealed. Products RT1184, RT1197, and Promote Dairy were used for alfalfa hay, whereas RT1181, RT1183, and Promote Dairy were used for corn silage. Groups of 30 bags, including six empty bags for correction, were placed upright in plastic containers, together with 150 mL of buffer (pH 6.0). The buffer used for this pretreatment was as described by Goering and Van Soest (1970) but without addition of reducing solution. Enzymes were added to the containers at the appropriate rates (1.5 mL/g forage DM), dissolved in 1 mL of distilled water, 20 h before addition of ruminal fluid. The mixtures were gently shaken to allow proper mixing and stored at room temperature (24°C). Ruminal fluid was collected from three lactating cows as described in Exp. 1. Four hundred milliliters of ruminal fluid was then added to each ANKOM fermentation jar, together with 1,600 mL of anaerobic buffer (adjusted to pH 6.0). Bags, plus all liquid contents in the plastic containers, were added to the fermentation jars, and fermentation allowed to continue for 96 h at 39°C. Bags were removed in quadruplicate (plus one empty bag per time point) at 0, 6, 18, 30, 48, and 96 h of incubation, and washed under cold tap water until excess water ran clear. Bags were dried at 55°C for 48 h, and DMD was determined. Fiber (NDF and ADF) degradation was determined sequentially on the same bags using the ANKOM200 fiber analysis system (ANKOM Corp.), following the procedures outlined by Van Soest et al. (1991). For the NDF analysis, α-amylase was included but sodium sulfite was excluded. After each analysis, bags were dried as described for DMD determination. The experiment was replicated on two occasions. Experiment 3. Results from Exp. 2 indicated that preparations RT1181 and RT1184 were effective in increasing the degradation of corn silage and alfalfa hay, respectively. It was of interest to determine whether these products would work on a mixture of those forages, and if they would work in combination. Therefore, an additional experiment was carried out with the following treatments: control (no enzyme), RT1181 alone, RT1184 alone, and a combination of RT1181 and RT1184 (1:1, vol/vol) at two final levels, 0.5 (8184 Low) or 1.5 (8184 High) mL/g forage DM. The forage used was a combination (1:1, wt/wt) of alfalfa hay and corn silage. Experimental protocols, determinations, and replications were identical to those described in Exp. 2. All donor animals used in the present study were cared for according to the Canadian Council on Animal Care guidelines (Ottawa, ON). Statistical Analyses Experiment 1 was a completely randomized design, with a model that included enzyme treatment and substrate as fixed effects. As a significant enzyme-substrate interaction was found, analyses were carried out separately for each forage source (alfalfa hay and corn silage). Differences among means were analyzed using the Mixed Procedures of SAS (SAS Inst. Inc., Cary, NC), with the PDIFF command invoked. Protein contents, total activities, and reducing sugars released were correlated to DMD values for each forage source using the Stepwise Regression Procedures of SAS. Data from Exp. 2 and 3 were analyzed as a completely randomized design with a factorial arrangement of treatments, using a model that included enzyme as fixed effect, and experimental run as a random effect. Unless stated otherwise, significance was declared at P < 0.05, whereas trends were discussed at P < 0.10. Results and Discussion Hypothesis Our hypothesis was that the in vitro degradation of forages would be correlated with one or more enzymic activities contained in the commercial enzyme products. In addition, it was hypothesized that the correlated enzymic activities would differ depending on the forage considered. Biochemical Characterization of Enzyme Products Protein content varied widely among the 23 enzyme products considered (Table 1). This was not surprising given the diversity of microbial sources, production procedures, and preservatives or carriers commonly used in the formulation of these products (Nieves et al., 1998). Because the contribution of the preservatives and carriers to the total protein contents in the enzyme products was unknown, the specific enzymic activities of the products are not presented here. Table 1. Protein concentrations (mg/mL), enzymic activities (μmol of sugar or ρ-nitrophenol min−1•g−1 or mm for protease), and reducing sugars released (mg) from the incubation of alfalfa hay (AH) and corn silage (CS) with the enzyme products     Enzymic activitya  RSb  Product  Protein  OSX  BX  CMC  SCELL  BG  XG  AGAL  LICH  AX  AMYL  LAM  ES  AF  GPY  XPY  PRT  GAPY  AH  CS  RT1180  114  222  143  591  32  535  317  0.0  832  108  14  0.0  8.6  0.7  11  3.0  7.3  2.8  1.2  0.7  RT1181  116  81  0.8  332  74  351  199  0.0  560  22  5.6  0.0  3.4  0.3  6.0  0.3  3.6  0.0  1.0  0.8  RT1182  37  3228  3160  72  0.0  6.4  34  0.0  57  915  7.6  0.0  3.4  0.4  4.5  5.0  7.2  1.0  0.7  0.2  RT1183  109  487  517  421  29  279  178  0.0  419  364  12  0.4  13  0.5  3.5  1.3  6.3  0.1  0.9  0.8  RT1184  128  28  6.7  0.0  0.5  53  2.1  0.0  71  2.6  7.9  0.0  12  0.0  0.0  0.0  29.6  0.0  0.5  0.0  RT1185  90  84  58  324  38  264  329  0.0  639  79  10  2.3  4.0  0.3  3.1  0.3  7.8  0.0  0.5  0.7  RT1186  120  429  336  665  15  526  242  0.0  647  177  10  31  3.6  0.6  15  3.2  0.0  1.8  0.7  1.2  RT1187  119  138  49  419  60  351  193  0.0  431  0.0  3.3  23  3.6  0.1  5.0  0.4  0.0  0.0  0.6  1.1  RT1188  121  145  47  356  26  310  132  0.0  345  0.0  15  10  8.0  0.4  4.3  0.9  8.4  0.0  0.4  0.6  RT1189  101  323  228  493  32  376  249  0.0  560  33  2.1  22  8.8  0.3  10  2.3  5.6  2.0  0.6  0.8  RT1190  467  1116  517  71  62  101  19  5.6  55  461  1586  199  25  0.1  10  0.3  30.1  6.3  11  1.5  RT1191  795  340  119  897  181  829  479  0.0  1464  87  28  69  11  1.0  12  0.4  0.0  0.0  10  3.9  RT1192  754  524  347  1047  123  875  774  0.0  1618  398  32  23  16  0.6  18  1.9  0.0  0.0  8.4  2.9  RT1193  259  74  17  88  0.0  16  38  0.0  32  132  283  129  6.0  1.2  4.3  3.3  21.0  2.2  5.6  0.4  RT1194  371  566  91  114  8.4  40  77  47  197  148  1892  138  3.1  0.1  2.0  0.1  24.5  5.1  3.6  0.7  RT1195  80  51  68  25  0.0  57  6.4  0.0  40  2.7  31  35  0.5  0.0  0.0  0.0  0.0  0.0  2.0  0.0  RT1196  315  2938  2063  178  0.0  710  42  0.0  790  857  1068  23  58  11  8.6  18  13.4  8.0  0.9  2.9  RT1197  546  2000  1466  713  164  1591  166  121  2076  871  694  174  103  3.7  58  13  16.7  1.9  0.6  2.2  RT1198  72  296  47  0.2  0.0  115  209  0.0  442  43  293  14  20  16  2.2  1.4  12.1  4.7  0.5  0.7  RT1199  65  95  85  75  66  5.6  23  190  46  66  9.3  119  26  5.2  1.4  0.0  8.6  13  2.1  0.4  RT1200  233  536  542  351  81  315  390  0.0  807  359  33  40  71  17  3.8  29  7.8  0.0  1.3  0.9  RT1201  179  2686  1945  21  0.2  4.8  0.0  0.0  16  518  465  82  0.0  0.0  0.0  0.0  7.4  0.0  1.7  2.7  Promotec  85  2729  2252  225  24  120  64  0.0  212  1236  9.1  43  7.5  0.5  15  0.6  20.0  0.0  ND  ND      Enzymic activitya  RSb  Product  Protein  OSX  BX  CMC  SCELL  BG  XG  AGAL  LICH  AX  AMYL  LAM  ES  AF  GPY  XPY  PRT  GAPY  AH  CS  RT1180  114  222  143  591  32  535  317  0.0  832  108  14  0.0  8.6  0.7  11  3.0  7.3  2.8  1.2  0.7  RT1181  116  81  0.8  332  74  351  199  0.0  560  22  5.6  0.0  3.4  0.3  6.0  0.3  3.6  0.0  1.0  0.8  RT1182  37  3228  3160  72  0.0  6.4  34  0.0  57  915  7.6  0.0  3.4  0.4  4.5  5.0  7.2  1.0  0.7  0.2  RT1183  109  487  517  421  29  279  178  0.0  419  364  12  0.4  13  0.5  3.5  1.3  6.3  0.1  0.9  0.8  RT1184  128  28  6.7  0.0  0.5  53  2.1  0.0  71  2.6  7.9  0.0  12  0.0  0.0  0.0  29.6  0.0  0.5  0.0  RT1185  90  84  58  324  38  264  329  0.0  639  79  10  2.3  4.0  0.3  3.1  0.3  7.8  0.0  0.5  0.7  RT1186  120  429  336  665  15  526  242  0.0  647  177  10  31  3.6  0.6  15  3.2  0.0  1.8  0.7  1.2  RT1187  119  138  49  419  60  351  193  0.0  431  0.0  3.3  23  3.6  0.1  5.0  0.4  0.0  0.0  0.6  1.1  RT1188  121  145  47  356  26  310  132  0.0  345  0.0  15  10  8.0  0.4  4.3  0.9  8.4  0.0  0.4  0.6  RT1189  101  323  228  493  32  376  249  0.0  560  33  2.1  22  8.8  0.3  10  2.3  5.6  2.0  0.6  0.8  RT1190  467  1116  517  71  62  101  19  5.6  55  461  1586  199  25  0.1  10  0.3  30.1  6.3  11  1.5  RT1191  795  340  119  897  181  829  479  0.0  1464  87  28  69  11  1.0  12  0.4  0.0  0.0  10  3.9  RT1192  754  524  347  1047  123  875  774  0.0  1618  398  32  23  16  0.6  18  1.9  0.0  0.0  8.4  2.9  RT1193  259  74  17  88  0.0  16  38  0.0  32  132  283  129  6.0  1.2  4.3  3.3  21.0  2.2  5.6  0.4  RT1194  371  566  91  114  8.4  40  77  47  197  148  1892  138  3.1  0.1  2.0  0.1  24.5  5.1  3.6  0.7  RT1195  80  51  68  25  0.0  57  6.4  0.0  40  2.7  31  35  0.5  0.0  0.0  0.0  0.0  0.0  2.0  0.0  RT1196  315  2938  2063  178  0.0  710  42  0.0  790  857  1068  23  58  11  8.6  18  13.4  8.0  0.9  2.9  RT1197  546  2000  1466  713  164  1591  166  121  2076  871  694  174  103  3.7  58  13  16.7  1.9  0.6  2.2  RT1198  72  296  47  0.2  0.0  115  209  0.0  442  43  293  14  20  16  2.2  1.4  12.1  4.7  0.5  0.7  RT1199  65  95  85  75  66  5.6  23  190  46  66  9.3  119  26  5.2  1.4  0.0  8.6  13  2.1  0.4  RT1200  233  536  542  351  81  315  390  0.0  807  359  33  40  71  17  3.8  29  7.8  0.0  1.3  0.9  RT1201  179  2686  1945  21  0.2  4.8  0.0  0.0  16  518  465  82  0.0  0.0  0.0  0.0  7.4  0.0  1.7  2.7  Promotec  85  2729  2252  225  24  120  64  0.0  212  1236  9.1  43  7.5  0.5  15  0.6  20.0  0.0  ND  ND  a OSX = xylanase (oat spelt); BX = xylanase (birchwood); CMC = endoglucanase; SCELL = exoglucanase; BG = β-glucanase; XG = xyloglucan; AGAL = arabinogalactan; LICH = lichenase; AX = arabinoxylan; AMYL = α-amylase; LAM = laminarinase; ES = esterase; AF = arabinofuranosidase; GPY = β-glucosidase; XPY = xylosidase; PRT = protease; GAPY = galactosidase. b RS = reducing sugars, determined after incubation of 25 mg of freeze-dried substrate at 39°C for 15 min with each enzyme product in triplicate. c Promote N.E.T. (Lot MO-E100). View Large Table 1. Protein concentrations (mg/mL), enzymic activities (μmol of sugar or ρ-nitrophenol min−1•g−1 or mm for protease), and reducing sugars released (mg) from the incubation of alfalfa hay (AH) and corn silage (CS) with the enzyme products     Enzymic activitya  RSb  Product  Protein  OSX  BX  CMC  SCELL  BG  XG  AGAL  LICH  AX  AMYL  LAM  ES  AF  GPY  XPY  PRT  GAPY  AH  CS  RT1180  114  222  143  591  32  535  317  0.0  832  108  14  0.0  8.6  0.7  11  3.0  7.3  2.8  1.2  0.7  RT1181  116  81  0.8  332  74  351  199  0.0  560  22  5.6  0.0  3.4  0.3  6.0  0.3  3.6  0.0  1.0  0.8  RT1182  37  3228  3160  72  0.0  6.4  34  0.0  57  915  7.6  0.0  3.4  0.4  4.5  5.0  7.2  1.0  0.7  0.2  RT1183  109  487  517  421  29  279  178  0.0  419  364  12  0.4  13  0.5  3.5  1.3  6.3  0.1  0.9  0.8  RT1184  128  28  6.7  0.0  0.5  53  2.1  0.0  71  2.6  7.9  0.0  12  0.0  0.0  0.0  29.6  0.0  0.5  0.0  RT1185  90  84  58  324  38  264  329  0.0  639  79  10  2.3  4.0  0.3  3.1  0.3  7.8  0.0  0.5  0.7  RT1186  120  429  336  665  15  526  242  0.0  647  177  10  31  3.6  0.6  15  3.2  0.0  1.8  0.7  1.2  RT1187  119  138  49  419  60  351  193  0.0  431  0.0  3.3  23  3.6  0.1  5.0  0.4  0.0  0.0  0.6  1.1  RT1188  121  145  47  356  26  310  132  0.0  345  0.0  15  10  8.0  0.4  4.3  0.9  8.4  0.0  0.4  0.6  RT1189  101  323  228  493  32  376  249  0.0  560  33  2.1  22  8.8  0.3  10  2.3  5.6  2.0  0.6  0.8  RT1190  467  1116  517  71  62  101  19  5.6  55  461  1586  199  25  0.1  10  0.3  30.1  6.3  11  1.5  RT1191  795  340  119  897  181  829  479  0.0  1464  87  28  69  11  1.0  12  0.4  0.0  0.0  10  3.9  RT1192  754  524  347  1047  123  875  774  0.0  1618  398  32  23  16  0.6  18  1.9  0.0  0.0  8.4  2.9  RT1193  259  74  17  88  0.0  16  38  0.0  32  132  283  129  6.0  1.2  4.3  3.3  21.0  2.2  5.6  0.4  RT1194  371  566  91  114  8.4  40  77  47  197  148  1892  138  3.1  0.1  2.0  0.1  24.5  5.1  3.6  0.7  RT1195  80  51  68  25  0.0  57  6.4  0.0  40  2.7  31  35  0.5  0.0  0.0  0.0  0.0  0.0  2.0  0.0  RT1196  315  2938  2063  178  0.0  710  42  0.0  790  857  1068  23  58  11  8.6  18  13.4  8.0  0.9  2.9  RT1197  546  2000  1466  713  164  1591  166  121  2076  871  694  174  103  3.7  58  13  16.7  1.9  0.6  2.2  RT1198  72  296  47  0.2  0.0  115  209  0.0  442  43  293  14  20  16  2.2  1.4  12.1  4.7  0.5  0.7  RT1199  65  95  85  75  66  5.6  23  190  46  66  9.3  119  26  5.2  1.4  0.0  8.6  13  2.1  0.4  RT1200  233  536  542  351  81  315  390  0.0  807  359  33  40  71  17  3.8  29  7.8  0.0  1.3  0.9  RT1201  179  2686  1945  21  0.2  4.8  0.0  0.0  16  518  465  82  0.0  0.0  0.0  0.0  7.4  0.0  1.7  2.7  Promotec  85  2729  2252  225  24  120  64  0.0  212  1236  9.1  43  7.5  0.5  15  0.6  20.0  0.0  ND  ND      Enzymic activitya  RSb  Product  Protein  OSX  BX  CMC  SCELL  BG  XG  AGAL  LICH  AX  AMYL  LAM  ES  AF  GPY  XPY  PRT  GAPY  AH  CS  RT1180  114  222  143  591  32  535  317  0.0  832  108  14  0.0  8.6  0.7  11  3.0  7.3  2.8  1.2  0.7  RT1181  116  81  0.8  332  74  351  199  0.0  560  22  5.6  0.0  3.4  0.3  6.0  0.3  3.6  0.0  1.0  0.8  RT1182  37  3228  3160  72  0.0  6.4  34  0.0  57  915  7.6  0.0  3.4  0.4  4.5  5.0  7.2  1.0  0.7  0.2  RT1183  109  487  517  421  29  279  178  0.0  419  364  12  0.4  13  0.5  3.5  1.3  6.3  0.1  0.9  0.8  RT1184  128  28  6.7  0.0  0.5  53  2.1  0.0  71  2.6  7.9  0.0  12  0.0  0.0  0.0  29.6  0.0  0.5  0.0  RT1185  90  84  58  324  38  264  329  0.0  639  79  10  2.3  4.0  0.3  3.1  0.3  7.8  0.0  0.5  0.7  RT1186  120  429  336  665  15  526  242  0.0  647  177  10  31  3.6  0.6  15  3.2  0.0  1.8  0.7  1.2  RT1187  119  138  49  419  60  351  193  0.0  431  0.0  3.3  23  3.6  0.1  5.0  0.4  0.0  0.0  0.6  1.1  RT1188  121  145  47  356  26  310  132  0.0  345  0.0  15  10  8.0  0.4  4.3  0.9  8.4  0.0  0.4  0.6  RT1189  101  323  228  493  32  376  249  0.0  560  33  2.1  22  8.8  0.3  10  2.3  5.6  2.0  0.6  0.8  RT1190  467  1116  517  71  62  101  19  5.6  55  461  1586  199  25  0.1  10  0.3  30.1  6.3  11  1.5  RT1191  795  340  119  897  181  829  479  0.0  1464  87  28  69  11  1.0  12  0.4  0.0  0.0  10  3.9  RT1192  754  524  347  1047  123  875  774  0.0  1618  398  32  23  16  0.6  18  1.9  0.0  0.0  8.4  2.9  RT1193  259  74  17  88  0.0  16  38  0.0  32  132  283  129  6.0  1.2  4.3  3.3  21.0  2.2  5.6  0.4  RT1194  371  566  91  114  8.4  40  77  47  197  148  1892  138  3.1  0.1  2.0  0.1  24.5  5.1  3.6  0.7  RT1195  80  51  68  25  0.0  57  6.4  0.0  40  2.7  31  35  0.5  0.0  0.0  0.0  0.0  0.0  2.0  0.0  RT1196  315  2938  2063  178  0.0  710  42  0.0  790  857  1068  23  58  11  8.6  18  13.4  8.0  0.9  2.9  RT1197  546  2000  1466  713  164  1591  166  121  2076  871  694  174  103  3.7  58  13  16.7  1.9  0.6  2.2  RT1198  72  296  47  0.2  0.0  115  209  0.0  442  43  293  14  20  16  2.2  1.4  12.1  4.7  0.5  0.7  RT1199  65  95  85  75  66  5.6  23  190  46  66  9.3  119  26  5.2  1.4  0.0  8.6  13  2.1  0.4  RT1200  233  536  542  351  81  315  390  0.0  807  359  33  40  71  17  3.8  29  7.8  0.0  1.3  0.9  RT1201  179  2686  1945  21  0.2  4.8  0.0  0.0  16  518  465  82  0.0  0.0  0.0  0.0  7.4  0.0  1.7  2.7  Promotec  85  2729  2252  225  24  120  64  0.0  212  1236  9.1  43  7.5  0.5  15  0.6  20.0  0.0  ND  ND  a OSX = xylanase (oat spelt); BX = xylanase (birchwood); CMC = endoglucanase; SCELL = exoglucanase; BG = β-glucanase; XG = xyloglucan; AGAL = arabinogalactan; LICH = lichenase; AX = arabinoxylan; AMYL = α-amylase; LAM = laminarinase; ES = esterase; AF = arabinofuranosidase; GPY = β-glucosidase; XPY = xylosidase; PRT = protease; GAPY = galactosidase. b RS = reducing sugars, determined after incubation of 25 mg of freeze-dried substrate at 39°C for 15 min with each enzyme product in triplicate. c Promote N.E.T. (Lot MO-E100). View Large All enzyme products showed a unique array of enzymic activities (Table 1). Enzyme activities were determined at physiological conditions, which generally differed from the optimal conditions reported for these enzymes (Bhat and Hazlewood, 2001). However, the conditions closely resembled the conditions under which the enzymes are expected to act. In the past, little attention was paid to this fact, and enzyme activities were sometimes determined at 50°C and at pH values as low as 4.8. Clearly, the applicability of these data is questionable (Kung, 2000). In terms of enzymic activities, RT1197 was the most concentrated of those tested, ranking within the first five preparations in 14 out of the 17 activities determined. Products RT1191, RT1192, RT1196, and RT1200 also showed high activities in general. Products RT1191, RT1192 and RT1197 were the most active against crystalline cellulose. Interestingly, several enzyme products showed considerable protease activity (e.g., RT1193, RT1194, RT1197, and Promote), even though most of them are marketed as “fibrolytic” preparations. An analysis of the release of reducing sugars from alfalfa hay and corn silage by the 22 enzyme candidates showed that RT1190, RT1191, and RT1192 were the most successful when both substrates were considered (Table 1). A stepwise regression of protein contents and enzyme activities on the release of reducing sugars showed that protein content alone explained 60% and 59% (P < 0.001) of the total variation for alfalfa hay and corn silage, respectively. Activity against β-glucan explained a further 24% (P < 0.001) of the variation in alfalfa hay, but its relationship with the release of reducing sugars was negative. In contrast, the release of reducing sugars from corn silage was positively correlated to activity against oat spelt xylan (P < 0.03), carboxymethylcellulose (P < 0.07), and crystalline cellulose (P < 0.05) but negatively correlated to activity against birchwood xylan (P < 0.01), starch (P < 0.001), and ρ-nitrophenyl-glucopyranoside (P < 0.003). Together, all these variables explained 96% of the total variation in the release of reducing sugars from corn silage (Table 2). The strong positive relationship between protein content and release of reducing sugars from both substrates may suggest that concentrated enzymes worked better, or at least faster, than more diluted samples, supplying enough enzyme activity to break down polysaccharides to simpler molecules in the short time allocated. The discrepancy in correlation between activity against oat spelt and birchwood xylan is puzzling because both activities are well related to each other (data not shown). Table 2. Relationship between enzymic activities and the release of reducing sugars from alfalfa hay and corn silage Forage  Variable  Partial R2  Model R2  F-value  P > F  Alfalfa hay  Protein content (x)  0.60    30.6  < 0.001    β-Glucanase (y)  0.24  0.84  28.3  < 0.001  Equation    Sugar = 0.017x − 0.029y + 0.2897      Corn silage  Protein content (a)  0.59    29.4  < 0.001    Xylanase (oat spelts) (b)  0.09  0.68  5.4  0.031    Endoglucanase (c)  0.05  0.74  3.8  0.067    Xylanase (birchwood) (d)  0.09  0.83  8.6  0.009    α-Amylase (e)  0.09  0.91  16.3  0.001    β-Glucosidase (f)  0.04  0.95  12.3  0.003    Exoglucanase (g)  0.01  0.96  4.8  0.047  Equation  Sugar = 0.001a + 0.027b + 0.009c − 0.026d − 0.005e − 0.032f + 0.025g − 0.2251  Forage  Variable  Partial R2  Model R2  F-value  P > F  Alfalfa hay  Protein content (x)  0.60    30.6  < 0.001    β-Glucanase (y)  0.24  0.84  28.3  < 0.001  Equation    Sugar = 0.017x − 0.029y + 0.2897      Corn silage  Protein content (a)  0.59    29.4  < 0.001    Xylanase (oat spelts) (b)  0.09  0.68  5.4  0.031    Endoglucanase (c)  0.05  0.74  3.8  0.067    Xylanase (birchwood) (d)  0.09  0.83  8.6  0.009    α-Amylase (e)  0.09  0.91  16.3  0.001    β-Glucosidase (f)  0.04  0.95  12.3  0.003    Exoglucanase (g)  0.01  0.96  4.8  0.047  Equation  Sugar = 0.001a + 0.027b + 0.009c − 0.026d − 0.005e − 0.032f + 0.025g − 0.2251  View Large Table 2. Relationship between enzymic activities and the release of reducing sugars from alfalfa hay and corn silage Forage  Variable  Partial R2  Model R2  F-value  P > F  Alfalfa hay  Protein content (x)  0.60    30.6  < 0.001    β-Glucanase (y)  0.24  0.84  28.3  < 0.001  Equation    Sugar = 0.017x − 0.029y + 0.2897      Corn silage  Protein content (a)  0.59    29.4  < 0.001    Xylanase (oat spelts) (b)  0.09  0.68  5.4  0.031    Endoglucanase (c)  0.05  0.74  3.8  0.067    Xylanase (birchwood) (d)  0.09  0.83  8.6  0.009    α-Amylase (e)  0.09  0.91  16.3  0.001    β-Glucosidase (f)  0.04  0.95  12.3  0.003    Exoglucanase (g)  0.01  0.96  4.8  0.047  Equation  Sugar = 0.001a + 0.027b + 0.009c − 0.026d − 0.005e − 0.032f + 0.025g − 0.2251  Forage  Variable  Partial R2  Model R2  F-value  P > F  Alfalfa hay  Protein content (x)  0.60    30.6  < 0.001    β-Glucanase (y)  0.24  0.84  28.3  < 0.001  Equation    Sugar = 0.017x − 0.029y + 0.2897      Corn silage  Protein content (a)  0.59    29.4  < 0.001    Xylanase (oat spelts) (b)  0.09  0.68  5.4  0.031    Endoglucanase (c)  0.05  0.74  3.8  0.067    Xylanase (birchwood) (d)  0.09  0.83  8.6  0.009    α-Amylase (e)  0.09  0.91  16.3  0.001    β-Glucosidase (f)  0.04  0.95  12.3  0.003    Exoglucanase (g)  0.01  0.96  4.8  0.047  Equation  Sugar = 0.001a + 0.027b + 0.009c − 0.026d − 0.005e − 0.032f + 0.025g − 0.2251  View Large In Vitro Degradation Assessment Experiment 1. Some of the products evaluated were effective in enhancing the hydrolytic potential of the ruminal fluid. With alfalfa hay as a substrate, five products (including Promote N.E.T.) increased (P < 0.05) DMD with respect to the untreated controls, after 18 h of incubation with ruminal fluid. Using corn silage, 11 products increased (P < 0.05) DMD, but Promote N.E.T. was not within this group. In contrast, PD and PB significantly increased (P < 0.05) corn silage DMD. Interestingly, the most effective enzymes against alfalfa hay were not the most effective ones against corn silage, suggesting a strong enzyme-feed specificity (Table 3). Enzyme-feed specificity is a well-known phenomenon and is believed to be one of the factors contributing to the observed inconsistencies in enzyme research (McAllister et al., 2001). Table 3. Effects of enzyme addition (1.5 μL/g of DM) on the apparent DMD (g/kg) of alfalfa hay or corn silage after 18 h of incubation with ruminal fluid Treatment  Alfalfa hay  Rankinga  Corn silage  Rankinga  Control  434.9  23  424.0  24  RT1180  450.4  18  438.0  19  RT1181  431.8  24  452.4c  7  RT1182  462.2  8  439.1  18  RT1183  459.4  10  462.7d  2  RT1184  477.4c  2  443.7  12  RT1185  454.3  15  441.6  16  RT1186  449.2  19  455.8d  5  RT1187  457.0  14  461.0d  3  RT1188  459.0  11  443.0  15  RT1189  454.3  16  454.8d  6  RT1190  472.0c  5  448.8c  10  RT1191  467.5  7  447.9c  11  RT1192  462.2  9  448.9c  9  RT1193  458.9  12  437.7  20  RT1194  443.5  22  432.9  21  RT1195  444.9  21  419.2  26  RT1196  475.4c  4  432.4  22  RT1197  468.8  6  423.8  25  RT1198  458.8  13  443.4  13  RT1199  452.9  17  449.0  8  RT1200  445.2  20  443.1  14  RT1201  479.7c  1  424.2  23  Promote N.E.T.  476.1c  3  439.6  17  Promote Dairy  NDd  ND  470.2d  1  Promote Beef  ND  ND  459.0d  4  SEM  24.56    7.96    Treatment  Alfalfa hay  Rankinga  Corn silage  Rankinga  Control  434.9  23  424.0  24  RT1180  450.4  18  438.0  19  RT1181  431.8  24  452.4c  7  RT1182  462.2  8  439.1  18  RT1183  459.4  10  462.7d  2  RT1184  477.4c  2  443.7  12  RT1185  454.3  15  441.6  16  RT1186  449.2  19  455.8d  5  RT1187  457.0  14  461.0d  3  RT1188  459.0  11  443.0  15  RT1189  454.3  16  454.8d  6  RT1190  472.0c  5  448.8c  10  RT1191  467.5  7  447.9c  11  RT1192  462.2  9  448.9c  9  RT1193  458.9  12  437.7  20  RT1194  443.5  22  432.9  21  RT1195  444.9  21  419.2  26  RT1196  475.4c  4  432.4  22  RT1197  468.8  6  423.8  25  RT1198  458.8  13  443.4  13  RT1199  452.9  17  449.0  8  RT1200  445.2  20  443.1  14  RT1201  479.7c  1  424.2  23  Promote N.E.T.  476.1c  3  439.6  17  Promote Dairy  NDd  ND  470.2d  1  Promote Beef  ND  ND  459.0d  4  SEM  24.56    7.96    a Relative ranking according to DMD. b ND = not determined. c,d Different from the control at P < 0.05 and P < 0.01, respectively. View Large Table 3. Effects of enzyme addition (1.5 μL/g of DM) on the apparent DMD (g/kg) of alfalfa hay or corn silage after 18 h of incubation with ruminal fluid Treatment  Alfalfa hay  Rankinga  Corn silage  Rankinga  Control  434.9  23  424.0  24  RT1180  450.4  18  438.0  19  RT1181  431.8  24  452.4c  7  RT1182  462.2  8  439.1  18  RT1183  459.4  10  462.7d  2  RT1184  477.4c  2  443.7  12  RT1185  454.3  15  441.6  16  RT1186  449.2  19  455.8d  5  RT1187  457.0  14  461.0d  3  RT1188  459.0  11  443.0  15  RT1189  454.3  16  454.8d  6  RT1190  472.0c  5  448.8c  10  RT1191  467.5  7  447.9c  11  RT1192  462.2  9  448.9c  9  RT1193  458.9  12  437.7  20  RT1194  443.5  22  432.9  21  RT1195  444.9  21  419.2  26  RT1196  475.4c  4  432.4  22  RT1197  468.8  6  423.8  25  RT1198  458.8  13  443.4  13  RT1199  452.9  17  449.0  8  RT1200  445.2  20  443.1  14  RT1201  479.7c  1  424.2  23  Promote N.E.T.  476.1c  3  439.6  17  Promote Dairy  NDd  ND  470.2d  1  Promote Beef  ND  ND  459.0d  4  SEM  24.56    7.96    Treatment  Alfalfa hay  Rankinga  Corn silage  Rankinga  Control  434.9  23  424.0  24  RT1180  450.4  18  438.0  19  RT1181  431.8  24  452.4c  7  RT1182  462.2  8  439.1  18  RT1183  459.4  10  462.7d  2  RT1184  477.4c  2  443.7  12  RT1185  454.3  15  441.6  16  RT1186  449.2  19  455.8d  5  RT1187  457.0  14  461.0d  3  RT1188  459.0  11  443.0  15  RT1189  454.3  16  454.8d  6  RT1190  472.0c  5  448.8c  10  RT1191  467.5  7  447.9c  11  RT1192  462.2  9  448.9c  9  RT1193  458.9  12  437.7  20  RT1194  443.5  22  432.9  21  RT1195  444.9  21  419.2  26  RT1196  475.4c  4  432.4  22  RT1197  468.8  6  423.8  25  RT1198  458.8  13  443.4  13  RT1199  452.9  17  449.0  8  RT1200  445.2  20  443.1  14  RT1201  479.7c  1  424.2  23  Promote N.E.T.  476.1c  3  439.6  17  Promote Dairy  NDd  ND  470.2d  1  Promote Beef  ND  ND  459.0d  4  SEM  24.56    7.96    a Relative ranking according to DMD. b ND = not determined. c,d Different from the control at P < 0.05 and P < 0.01, respectively. View Large When a stepwise multiple regression of protein concentrations, total enzyme activities, and reducing sugars release with in vitro ruminal degradation values was performed, a positive (P = 0.01) correlation between xylanase (oat spelt) and alfalfa DMD was observed. Protease activity was also positively related (P < 0.10) with alfalfa DMD. However, the proportion of the variance explained by the model was less than 40%. Activity against oat spelt xylan was also significant for corn silage (P = 0.04), but the nature of the relationship was negative (Table 4). It is unclear, however, whether this negative correlation indicates a cause and effect relationship between low xylanase activity and high DMD in corn silage. Nsereko et al. (2000) reported a positive correlation between xylanase activity and the degradation of alfalfa NDF in vitro, which they attributed to a modification of the fiber structure by enzyme action. In agreement with these data, Vanbelle and Bertin (1989) found that enzymes having high C5-producing activity (xylose or arabinose) enhanced the in vitro degradation of alfalfa. Furthermore, Nsereko et al. (2000) reported negative correlations between β-linked glucan hydrolases (including cellulase) and alfalfa fiber degradation. The mechanisms for this apparent inhibition are not known but could include blockage of enzyme binding sites (Nsereko et al. 2000), a decrease in the microbial attachment to feeds (Morgavi et al. 2000a), or a direct inhibitory effect over the production of microbial enzymes from ruminal microorganisms. Table 4. Relationship between enzymic activities (μmol of xylose min−1•g−1) and the apparent DMD (g/kg) of alfalfa hay and corn silage, after 18 h of incubation with ruminal fluid Forage  Enzymic activity  Regression equation  Partial R2  Model R2  P > F  Alfalfa hay  Xylanase (oat spelts) (x)    0.29    0.010    Protease (y)    0.10  0.39  0.096      DMD = 0.04x + 0.41y + 449.9        Corn silage  Xylanase (oat spelts)  DMD = −0.033x + 446.6  —  0.19  0.044  Forage  Enzymic activity  Regression equation  Partial R2  Model R2  P > F  Alfalfa hay  Xylanase (oat spelts) (x)    0.29    0.010    Protease (y)    0.10  0.39  0.096      DMD = 0.04x + 0.41y + 449.9        Corn silage  Xylanase (oat spelts)  DMD = −0.033x + 446.6  —  0.19  0.044  View Large Table 4. Relationship between enzymic activities (μmol of xylose min−1•g−1) and the apparent DMD (g/kg) of alfalfa hay and corn silage, after 18 h of incubation with ruminal fluid Forage  Enzymic activity  Regression equation  Partial R2  Model R2  P > F  Alfalfa hay  Xylanase (oat spelts) (x)    0.29    0.010    Protease (y)    0.10  0.39  0.096      DMD = 0.04x + 0.41y + 449.9        Corn silage  Xylanase (oat spelts)  DMD = −0.033x + 446.6  —  0.19  0.044  Forage  Enzymic activity  Regression equation  Partial R2  Model R2  P > F  Alfalfa hay  Xylanase (oat spelts) (x)    0.29    0.010    Protease (y)    0.10  0.39  0.096      DMD = 0.04x + 0.41y + 449.9        Corn silage  Xylanase (oat spelts)  DMD = −0.033x + 446.6  —  0.19  0.044  View Large Grabber et al. (2002) reported that the degradation of xylans from alfalfa cell walls was severely restricted when compared to that of other polysaccharides, probably as a result of crosslinks involving lignin. This suggests that xylanase alone might be ineffective if not accompanied by other enzymes capable of cleaving these cross-links. Unlike grasses, ferulic acid does not appear to be involved in the interactions between xylans and lignin in alfalfa (Grabber et al., 2002), thus indicating that other chemical structures are present. It has been suggested (Jung, 1997), that tyrosine residues could play a role in the cross-linking of dicotyledonous plants; however, the specific mechanisms remain elusive (Iiyama et al., 1994; Ringli et al., 2001). The simultaneous correlation between xylanase and protease activities and alfalfa DMD seems to support, at least in part, this theory. In addition, factors such as accessibility (Jung et al., 2000) or the presence of debranching enzymes (Greve et al., 1984) also appear to have an impact on alfalfa cell wall degradation. When corn silage was considered, no correlations other than those already mentioned (i.e., negative relationship with xylanase) were found. Other authors (Wallace et al., 2001) have suggested a possible limiting role for endoglucanase activity on corn silage in vitro fermentation, which was not found in our study. The fact that enzyme activities explained only about a third of the in vitro degradation should not be taken to mean that biochemical characterization of enzymes before use is not important. Other enzymic activities not examined in this study (e.g., pectinases, α-glucuronidase) might be important. Random use of enzymes in ruminant diets will only discourage or delay their development (Officer, 2000; McAllister et al., 2001). A thorough characterization of products found to be consistently effective should lead to the development of better, science-based enzyme additives. In addition, the strong relationship found between enzyme activities and the release of reducing sugars may be of importance for other applications, such as the use of enzymes at ensiling. Products with higher hydrolytic capacity would be expected to be more effective in enhancing the silage fermentation process, and the presence of undesirable enzymic activities would be detected before the product is added to the forage. Colombatto et al. (2001) showed that a commercial product marketed as a hemicellulase-rich enzyme also contained substantial amounts of α-amylase activities, which had negative nutritional consequences when added to forage corn at ensiling. Experiment 2. Products RT1184 and RT1197 were selected for further studies in alfalfa hay, whereas RT1181 and RT1183 were selected for corn silage. In addition, PD was included for both forages. The ANKOM system was used to determine DM and fiber degradation kinetics. Confirming our previous results, product RT1184 increased (P < 0.05) the degradation of alfalfa hay at 6 h (+9.0%), with a trend (P < 0.10) toward improving the degradation at 0 h (8.8%; Table 5). No differences were detected after 6 h of incubation for any of the treatments in alfalfa. In corn silage, product RT1181 increased (P < 0.05) DMD after 6 h of incubation and tended to increase (P < 0.10) DMD at 30 h. In addition, all enzyme products (RT1181, RT1183, and PD) increased (P < 0.05) 48-h DMD (Table 5). The latter is surprising given the general agreement that enzymes increase the rate, but not extent, of degradation (Colombatto, 2000; Beauchemin et al., 2001). However, 48-h DMD was not an endpoint for corn silage, as considerable degradation still took place after this time (between 10 and 14 percentage units). This is consistent with earlier observations by Wilson and Mertens (1995), who reported that a large proportion of corn's potentially degradable cell walls are composed of thick secondary cell walls, which are slowly degraded. Therefore, it is likely that active degradation was still under way during the 30- to 48-h incubation period, in contrast to what was observed in alfalfa hay. Table 5. Dry matter degradation (g/kg) of alfalfa hay or corn silage, untreated or treated with enzyme products at 1.5 μL/g of DM   Incubation time, h  Treatment  0  6  18  30  48  96  ——— Alfalfa hay ———  Control  307.0  409.6c  575.9  690.6  745.2  765.9  Promote Dairy  313.3  386.8c  573.0  680.2  741.9  762.6  RT1184  334.0  446.3d  609.0  689.4  744.8  769.7  RT1197  319.2  410.6c  568.8  680.2  739.2  769.9  SEMa  14.14  23.13  25.08  24.19  11.06  7.53  ——— Corn silage ———  Control  294.1  318.2c  455.3cd  521.6  619.2c  764.3cd  Promote Dairy  288.1  322.7cd  435.9c  523.4  648.7d  754.8cd  RT1181  307.9  344.5d  476.4d  549.3  641.6d  767.1d  RT1183  279.7  318.7c  451.6cd  527.5  634.7d  752.8c  SEM  29.79  38.61  29.09  24.39  10.14  13.05    Incubation time, h  Treatment  0  6  18  30  48  96  ——— Alfalfa hay ———  Control  307.0  409.6c  575.9  690.6  745.2  765.9  Promote Dairy  313.3  386.8c  573.0  680.2  741.9  762.6  RT1184  334.0  446.3d  609.0  689.4  744.8  769.7  RT1197  319.2  410.6c  568.8  680.2  739.2  769.9  SEMa  14.14  23.13  25.08  24.19  11.06  7.53  ——— Corn silage ———  Control  294.1  318.2c  455.3cd  521.6  619.2c  764.3cd  Promote Dairy  288.1  322.7cd  435.9c  523.4  648.7d  754.8cd  RT1181  307.9  344.5d  476.4d  549.3  641.6d  767.1d  RT1183  279.7  318.7c  451.6cd  527.5  634.7d  752.8c  SEM  29.79  38.61  29.09  24.39  10.14  13.05  a SEM = Standard error of the treatment means, n = 2. c,d Within substrates and columns, means without common superscripts differ (P < 0.05). View Large Table 5. Dry matter degradation (g/kg) of alfalfa hay or corn silage, untreated or treated with enzyme products at 1.5 μL/g of DM   Incubation time, h  Treatment  0  6  18  30  48  96  ——— Alfalfa hay ———  Control  307.0  409.6c  575.9  690.6  745.2  765.9  Promote Dairy  313.3  386.8c  573.0  680.2  741.9  762.6  RT1184  334.0  446.3d  609.0  689.4  744.8  769.7  RT1197  319.2  410.6c  568.8  680.2  739.2  769.9  SEMa  14.14  23.13  25.08  24.19  11.06  7.53  ——— Corn silage ———  Control  294.1  318.2c  455.3cd  521.6  619.2c  764.3cd  Promote Dairy  288.1  322.7cd  435.9c  523.4  648.7d  754.8cd  RT1181  307.9  344.5d  476.4d  549.3  641.6d  767.1d  RT1183  279.7  318.7c  451.6cd  527.5  634.7d  752.8c  SEM  29.79  38.61  29.09  24.39  10.14  13.05    Incubation time, h  Treatment  0  6  18  30  48  96  ——— Alfalfa hay ———  Control  307.0  409.6c  575.9  690.6  745.2  765.9  Promote Dairy  313.3  386.8c  573.0  680.2  741.9  762.6  RT1184  334.0  446.3d  609.0  689.4  744.8  769.7  RT1197  319.2  410.6c  568.8  680.2  739.2  769.9  SEMa  14.14  23.13  25.08  24.19  11.06  7.53  ——— Corn silage ———  Control  294.1  318.2c  455.3cd  521.6  619.2c  764.3cd  Promote Dairy  288.1  322.7cd  435.9c  523.4  648.7d  754.8cd  RT1181  307.9  344.5d  476.4d  549.3  641.6d  767.1d  RT1183  279.7  318.7c  451.6cd  527.5  634.7d  752.8c  SEM  29.79  38.61  29.09  24.39  10.14  13.05  a SEM = Standard error of the treatment means, n = 2. c,d Within substrates and columns, means without common superscripts differ (P < 0.05). View Large The fiber (NDF, ADF, and hemicellulose) degradation kinetics are shown in Table 6 (alfalfa hay) and Table 7 (corn silage). In agreement with our previous data, RT1184 increased (P < 0.05) the hemicellulose degradation of the alfalfa hay at 6 h of incubation almost by 100%, whereas sizeable increases (albeit nonsignificant) were observed in NDF degradation after 6 and 18 h of incubation for the same enzyme treatment. In contrast, product RT1197 failed to show differences with respect to the control. It is evident that most of the available fiber had been degraded by 48 h and that enzymes merely increased the rate of degradation. The fact that very little of the fiber fraction was degraded at 0 h, coupled with the increased hemicellulose degradation after 6 h, strongly suggests that RT1184 removed some components that presented a physical barrier to degradation. The fact that RT1184 contains mainly protease activity (Table 1) may suggest that protein is the component being removed. Table 6. Fiber degradation of alfalfa hay, untreated or treated with enzyme products at 1.5 μL/g of DM   Incubation time, h  Treatment  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  −12.3  34.0cd  217.6  365.8  454.6  493.0  Promote Dairy  5.8  19.6c  188.2  343.6  452.0  490.7  RT1184  12.3  51.3d  240.1  354.9  449.8  509.0  RT1197  −4.7  53.7d  198.0  343.3  454.0  500.8  SEMa  27.25  22.53  38.01  33.27  22.68  16.37  ——— ADF, g/kg ———  Control  −8.8  −3.0  153.5  322.4  408.0  440.5  Promote Dairy  0.1  −25.9  116.3  289.9  407.3  436.3  RT1184  12.2  −20.2  167.4  303.4  407.4  452.9  RT1197  −7.5  16.6  130.2  300.3  417.0  461.0  SEM  34.36  27.12  35.35  36.63  23.04  19.30  ——— Hemicellulose, g/kg ———  Control  −19.2  106.1c  342.6  450.4  545.5  595.1  Promote Dairy  17.1  108.4c  328.4  448.2  539.0  596.4  RT1184  12.5  190.8d  381.9  455.2  532.5  606.1  RT1197  0.6  125.8cd  329.9  426.9  526.1  578.3  SEM  20.41  23.55  45.47  29.59  24.79  16.62    Incubation time, h  Treatment  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  −12.3  34.0cd  217.6  365.8  454.6  493.0  Promote Dairy  5.8  19.6c  188.2  343.6  452.0  490.7  RT1184  12.3  51.3d  240.1  354.9  449.8  509.0  RT1197  −4.7  53.7d  198.0  343.3  454.0  500.8  SEMa  27.25  22.53  38.01  33.27  22.68  16.37  ——— ADF, g/kg ———  Control  −8.8  −3.0  153.5  322.4  408.0  440.5  Promote Dairy  0.1  −25.9  116.3  289.9  407.3  436.3  RT1184  12.2  −20.2  167.4  303.4  407.4  452.9  RT1197  −7.5  16.6  130.2  300.3  417.0  461.0  SEM  34.36  27.12  35.35  36.63  23.04  19.30  ——— Hemicellulose, g/kg ———  Control  −19.2  106.1c  342.6  450.4  545.5  595.1  Promote Dairy  17.1  108.4c  328.4  448.2  539.0  596.4  RT1184  12.5  190.8d  381.9  455.2  532.5  606.1  RT1197  0.6  125.8cd  329.9  426.9  526.1  578.3  SEM  20.41  23.55  45.47  29.59  24.79  16.62  a SEM = standard error of the treatment means, n = 2. c,d Within fractions and columns, means without common superscripts differ (P < 0.05). View Large Table 6. Fiber degradation of alfalfa hay, untreated or treated with enzyme products at 1.5 μL/g of DM   Incubation time, h  Treatment  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  −12.3  34.0cd  217.6  365.8  454.6  493.0  Promote Dairy  5.8  19.6c  188.2  343.6  452.0  490.7  RT1184  12.3  51.3d  240.1  354.9  449.8  509.0  RT1197  −4.7  53.7d  198.0  343.3  454.0  500.8  SEMa  27.25  22.53  38.01  33.27  22.68  16.37  ——— ADF, g/kg ———  Control  −8.8  −3.0  153.5  322.4  408.0  440.5  Promote Dairy  0.1  −25.9  116.3  289.9  407.3  436.3  RT1184  12.2  −20.2  167.4  303.4  407.4  452.9  RT1197  −7.5  16.6  130.2  300.3  417.0  461.0  SEM  34.36  27.12  35.35  36.63  23.04  19.30  ——— Hemicellulose, g/kg ———  Control  −19.2  106.1c  342.6  450.4  545.5  595.1  Promote Dairy  17.1  108.4c  328.4  448.2  539.0  596.4  RT1184  12.5  190.8d  381.9  455.2  532.5  606.1  RT1197  0.6  125.8cd  329.9  426.9  526.1  578.3  SEM  20.41  23.55  45.47  29.59  24.79  16.62    Incubation time, h  Treatment  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  −12.3  34.0cd  217.6  365.8  454.6  493.0  Promote Dairy  5.8  19.6c  188.2  343.6  452.0  490.7  RT1184  12.3  51.3d  240.1  354.9  449.8  509.0  RT1197  −4.7  53.7d  198.0  343.3  454.0  500.8  SEMa  27.25  22.53  38.01  33.27  22.68  16.37  ——— ADF, g/kg ———  Control  −8.8  −3.0  153.5  322.4  408.0  440.5  Promote Dairy  0.1  −25.9  116.3  289.9  407.3  436.3  RT1184  12.2  −20.2  167.4  303.4  407.4  452.9  RT1197  −7.5  16.6  130.2  300.3  417.0  461.0  SEM  34.36  27.12  35.35  36.63  23.04  19.30  ——— Hemicellulose, g/kg ———  Control  −19.2  106.1c  342.6  450.4  545.5  595.1  Promote Dairy  17.1  108.4c  328.4  448.2  539.0  596.4  RT1184  12.5  190.8d  381.9  455.2  532.5  606.1  RT1197  0.6  125.8cd  329.9  426.9  526.1  578.3  SEM  20.41  23.55  45.47  29.59  24.79  16.62  a SEM = standard error of the treatment means, n = 2. c,d Within fractions and columns, means without common superscripts differ (P < 0.05). View Large Table 7. Fiber degradation of corn silage, untreated or treated with enzyme products at 1.5 μL/g of DM   Incubation time, h  Treatment  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  5.7  17.5  116.1c  193.5  327.3c  581.0cd  Promote Dairy  29.2  39.4  119.3c  196.6  380.0d  560.3d  RT1181  44.3  45.3  147.0d  225.7  368.7d  587.5d  RT1183  18.2  17.7  104.2c  184.2  354.6cd  565.1c  SEMa  14.88  12.53  19.26  15.74  13.75  19.85  ——— ADF, g/kg ———  Control  −4.2  14.9  78.0c  153.6  292.1c  553.7  Promote Dairy  13.3  21.6  79.7c  159.1  345.6d  527.6  RT1181  42.0  48.2  111.7d  204.6  333.5d  554.8  RT1183  4.6  17.1  72.5c  154.8  321.5cd  541.8  SEM  19.85  23.05  12.99  17.56  12.97  27.19  ——— Hemicellulose, g/kg ———  Control  17.6  20.5c  161.5d  241.1  369.1c  613.7de  Promote Dairy  48.1  60.6e  166.4d  241.4  421.0d  599.3cd  RT1181  47.1  41.8d  189.0d  250.8  410.6cd  626.4e  RT1183  34.4  18.4c  142.0c  219.2  393.8cd  592.8c  SEM  10.27  2.39  27.12  14.44  15.63  12.15    Incubation time, h  Treatment  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  5.7  17.5  116.1c  193.5  327.3c  581.0cd  Promote Dairy  29.2  39.4  119.3c  196.6  380.0d  560.3d  RT1181  44.3  45.3  147.0d  225.7  368.7d  587.5d  RT1183  18.2  17.7  104.2c  184.2  354.6cd  565.1c  SEMa  14.88  12.53  19.26  15.74  13.75  19.85  ——— ADF, g/kg ———  Control  −4.2  14.9  78.0c  153.6  292.1c  553.7  Promote Dairy  13.3  21.6  79.7c  159.1  345.6d  527.6  RT1181  42.0  48.2  111.7d  204.6  333.5d  554.8  RT1183  4.6  17.1  72.5c  154.8  321.5cd  541.8  SEM  19.85  23.05  12.99  17.56  12.97  27.19  ——— Hemicellulose, g/kg ———  Control  17.6  20.5c  161.5d  241.1  369.1c  613.7de  Promote Dairy  48.1  60.6e  166.4d  241.4  421.0d  599.3cd  RT1181  47.1  41.8d  189.0d  250.8  410.6cd  626.4e  RT1183  34.4  18.4c  142.0c  219.2  393.8cd  592.8c  SEM  10.27  2.39  27.12  14.44  15.63  12.15  a SEM = standard error of the treatment means, n = 2. c,d,e Within fractions and columns, means without common superscripts differ (P < 0.05). View Large Table 7. Fiber degradation of corn silage, untreated or treated with enzyme products at 1.5 μL/g of DM   Incubation time, h  Treatment  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  5.7  17.5  116.1c  193.5  327.3c  581.0cd  Promote Dairy  29.2  39.4  119.3c  196.6  380.0d  560.3d  RT1181  44.3  45.3  147.0d  225.7  368.7d  587.5d  RT1183  18.2  17.7  104.2c  184.2  354.6cd  565.1c  SEMa  14.88  12.53  19.26  15.74  13.75  19.85  ——— ADF, g/kg ———  Control  −4.2  14.9  78.0c  153.6  292.1c  553.7  Promote Dairy  13.3  21.6  79.7c  159.1  345.6d  527.6  RT1181  42.0  48.2  111.7d  204.6  333.5d  554.8  RT1183  4.6  17.1  72.5c  154.8  321.5cd  541.8  SEM  19.85  23.05  12.99  17.56  12.97  27.19  ——— Hemicellulose, g/kg ———  Control  17.6  20.5c  161.5d  241.1  369.1c  613.7de  Promote Dairy  48.1  60.6e  166.4d  241.4  421.0d  599.3cd  RT1181  47.1  41.8d  189.0d  250.8  410.6cd  626.4e  RT1183  34.4  18.4c  142.0c  219.2  393.8cd  592.8c  SEM  10.27  2.39  27.12  14.44  15.63  12.15    Incubation time, h  Treatment  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  5.7  17.5  116.1c  193.5  327.3c  581.0cd  Promote Dairy  29.2  39.4  119.3c  196.6  380.0d  560.3d  RT1181  44.3  45.3  147.0d  225.7  368.7d  587.5d  RT1183  18.2  17.7  104.2c  184.2  354.6cd  565.1c  SEMa  14.88  12.53  19.26  15.74  13.75  19.85  ——— ADF, g/kg ———  Control  −4.2  14.9  78.0c  153.6  292.1c  553.7  Promote Dairy  13.3  21.6  79.7c  159.1  345.6d  527.6  RT1181  42.0  48.2  111.7d  204.6  333.5d  554.8  RT1183  4.6  17.1  72.5c  154.8  321.5cd  541.8  SEM  19.85  23.05  12.99  17.56  12.97  27.19  ——— Hemicellulose, g/kg ———  Control  17.6  20.5c  161.5d  241.1  369.1c  613.7de  Promote Dairy  48.1  60.6e  166.4d  241.4  421.0d  599.3cd  RT1181  47.1  41.8d  189.0d  250.8  410.6cd  626.4e  RT1183  34.4  18.4c  142.0c  219.2  393.8cd  592.8c  SEM  10.27  2.39  27.12  14.44  15.63  12.15  a SEM = standard error of the treatment means, n = 2. c,d,e Within fractions and columns, means without common superscripts differ (P < 0.05). View Large In corn silage, product RT1181 increased NDF and ADF degradation at all times up to 48 h of incubation, with the values achieving significance (P < 0.05) at h 18 and 48 (Table 7). Hemicellulose degradation was increased (P < 0.05) by the same enzyme at 6 h incubation, and tended to be higher (P < 0.10) than the controls at h 18 (+17%) and 48 (11%). Promote Dairy increased (P < 0.05) NDF, ADF, and hemicellulose degradation at 48 h of incubation, with respect to the controls (Table 7). In addition, hemicellulose degradation was increased (P < 0.05) at 6 h of incubation in the PD treatments. In contrast to what was found with alfalfa hay, there was no indication of “preingestive” effects (i.e., 0-h differences) between the controls and any of the enzyme treatments. This finding suggests that, with corn silage, the enzyme products worked only at the ruminal level, in contrast to Wallace et al. (2001), who suggested that preruminal interactions were more important than ruminal interactions for maximizing the efficacy of enzyme additives for ruminants. However, different enzyme products used in various studies make direct comparisons difficult. Evidence suggests that alfalfa is benefited by a pretreatment period, possibly due to small structural changes to the cell wall (Nsereko et al., 2000), whereas the situation with corn silage is unclear. Wang et al. (2002) found that a concentrated enzyme product enhanced the release of reducing sugars from corn silage, encouraging the growth of epiphytic bacteria, which may accelerate aerobic decomposition of the silage. Therefore, it appears that the optimal length of an enzyme-feed interaction time before feeding may depend on the forage under study. The degradation of the nonfibrous fraction was calculated to determine the proportion of the increase in DMD attributable to the fiber fraction. Results for both forages are shown in Table 8. Fiber degradation explained about a third of the DMD during the first 18 h of incubation, when product RT1184 was added to alfalfa. When product RT1181 was added to corn silage, fiber degradation contributed to at least 50% of the total increase in degradation, with the significant increases in DMD found at 48 h being almost totally explained (86.4%) by an increase in fiber degradation. These findings further confirm that products RT1181 and RT1184 have different modes of action. It seems that RT1181, an enzyme product derived from Trichoderma longibrachiatum, concentrates its action on the fiber once in the in vitro ruminal system, which is consistent with a synergistic interaction between enzymes derived from T. longibrachiatum and ruminal enzymes described by Morgavi et al. (2000b) using the same substrate. In turn, RT1184, a product derived from Bacillus spp., acts mainly on the nonfibrous fraction (possibly protein), and the effects are evident at h 0 of incubation, which may indicate the removal of structural barriers that retard microbial colonization and degradation of alfalfa, as suggested by Nsereko et al. (2000). Table 8. Degradation profiles (g/kg DM) of the nonfiber fractions (100 minus NDF), and percentage of increase in DMD for treatments RT1184 and RT1181 attributable to NDF degradation     Incubation time, h  Substrate  Treatment  0  6  18  30  48  96  Alfalfa hay    Control  311.7  396.6  492.8  550.9  571.5  577.6    Promote Dairy  311.1  379.3  501.1  548.9  569.2  575.2    RT1184  329.3  426.7  517.3  553.8  573.0  575.3    RT1197  321.0  390.1  493.2  549.1  565.8  578.6      Increase in DMD, %    8.8  9.0  5.7  −0.2  −0.05  0.5      Increase in DMD due to NDF, %    34.8  18.0  25.9  0  0  100  Corn silage    Control  291.4  310.0  401.0  431.2  466.2  492.7    Promote Dairy  274.5  304.3  380.1  431.5  471.1  492.9    RT1181  287.2  323.3  407.7  443.8  469.3  492.5    RT1183  271.2  310.4  402.9  441.4  469.0  488.7      Increase in DMD, %    4.7  8.3  4.6  5.3  3.6  0.4      Increase in DMD due to NDF, %    100  49.4  68.4  54.3  86.4  100      Incubation time, h  Substrate  Treatment  0  6  18  30  48  96  Alfalfa hay    Control  311.7  396.6  492.8  550.9  571.5  577.6    Promote Dairy  311.1  379.3  501.1  548.9  569.2  575.2    RT1184  329.3  426.7  517.3  553.8  573.0  575.3    RT1197  321.0  390.1  493.2  549.1  565.8  578.6      Increase in DMD, %    8.8  9.0  5.7  −0.2  −0.05  0.5      Increase in DMD due to NDF, %    34.8  18.0  25.9  0  0  100  Corn silage    Control  291.4  310.0  401.0  431.2  466.2  492.7    Promote Dairy  274.5  304.3  380.1  431.5  471.1  492.9    RT1181  287.2  323.3  407.7  443.8  469.3  492.5    RT1183  271.2  310.4  402.9  441.4  469.0  488.7      Increase in DMD, %    4.7  8.3  4.6  5.3  3.6  0.4      Increase in DMD due to NDF, %    100  49.4  68.4  54.3  86.4  100  View Large Table 8. Degradation profiles (g/kg DM) of the nonfiber fractions (100 minus NDF), and percentage of increase in DMD for treatments RT1184 and RT1181 attributable to NDF degradation     Incubation time, h  Substrate  Treatment  0  6  18  30  48  96  Alfalfa hay    Control  311.7  396.6  492.8  550.9  571.5  577.6    Promote Dairy  311.1  379.3  501.1  548.9  569.2  575.2    RT1184  329.3  426.7  517.3  553.8  573.0  575.3    RT1197  321.0  390.1  493.2  549.1  565.8  578.6      Increase in DMD, %    8.8  9.0  5.7  −0.2  −0.05  0.5      Increase in DMD due to NDF, %    34.8  18.0  25.9  0  0  100  Corn silage    Control  291.4  310.0  401.0  431.2  466.2  492.7    Promote Dairy  274.5  304.3  380.1  431.5  471.1  492.9    RT1181  287.2  323.3  407.7  443.8  469.3  492.5    RT1183  271.2  310.4  402.9  441.4  469.0  488.7      Increase in DMD, %    4.7  8.3  4.6  5.3  3.6  0.4      Increase in DMD due to NDF, %    100  49.4  68.4  54.3  86.4  100      Incubation time, h  Substrate  Treatment  0  6  18  30  48  96  Alfalfa hay    Control  311.7  396.6  492.8  550.9  571.5  577.6    Promote Dairy  311.1  379.3  501.1  548.9  569.2  575.2    RT1184  329.3  426.7  517.3  553.8  573.0  575.3    RT1197  321.0  390.1  493.2  549.1  565.8  578.6      Increase in DMD, %    8.8  9.0  5.7  −0.2  −0.05  0.5      Increase in DMD due to NDF, %    34.8  18.0  25.9  0  0  100  Corn silage    Control  291.4  310.0  401.0  431.2  466.2  492.7    Promote Dairy  274.5  304.3  380.1  431.5  471.1  492.9    RT1181  287.2  323.3  407.7  443.8  469.3  492.5    RT1183  271.2  310.4  402.9  441.4  469.0  488.7      Increase in DMD, %    4.7  8.3  4.6  5.3  3.6  0.4      Increase in DMD due to NDF, %    100  49.4  68.4  54.3  86.4  100  View Large Clearly, development of enzyme mixtures specific for only one type of forage (e.g., alfalfa) is unlikely to be attractive for feed companies, or even for producers, as they usually work with combinations of forages, grains, by-products, and so on. Therefore, development of enzymes effective for application to feed combinations should be one of the goals of any enzyme research program. Experiment 3. The last phase of studies examined the effects of two enzyme products (RT1181 and RT1184), alone or in combination (denoted 8184), on the kinetics of degradation of a combination of alfalfa hay and corn silage (1:1 wt/wt), using the ANKOM fermentation system. The results for DMD are shown in Table 9. In agreement with Exp. 2, RT1184 increased (P < 0.05) DMD of the alfalfa-corn silage combination at h 6 and 18 of incubation. It also increased (P < 0.05) DMD at h 0, indicating the presence of “preingestive” effects. Moreover, the degree of improvement with respect to the controls remained fairly constant between h 0 and 18, which suggests that the improvement at h 0 was not achieved at the expense of the most readily digestible fractions (i.e., those degraded within the first 12 h of incubation). That would have been the case had the degradability at h 6 or 18 been equal to that of the controls. Evidence suggests that degradation rate started to slow down between h 18 and 30 incubation, consistent with the time at which fiber fractions are attacked by ruminal microbes when incubated in vitro. Analysis of the fiber degradation in the RT1184 treatment indicated that the increase in DMD was accompanied by an increase (P < 0.05) in NDF degradation at h 6 and a trend (P < 0.10) toward an increase in NDF degradation at h 18, and an increase in hemicellulose degradation at h 6 and 18 (Table 10). Table 9. Dry matter degradation (g/kg) of a mixture of alfalfa hay and corn silage, untreated or treated with enzyme products   Incubation time, h  Treatmenta  0  6  18  30  48  96  Control  319.1c  411.1c  563.4c  632.8  709.4  774.5c  RT1181  322.0c  424.3cd  564.3c  649.5  724.6  774.9c  RT1184  354.4d  445.2d  592.4d  657.2  739.8  782.9d  8184 Low  336.2cd  410.8c  55.89d  637.1  720.1  781.4d  8184 High  336.8cd  442.0cd  579.4cd  655.5  733.9  783.4d  SEMb  12.91  11.67  7.83  10.55  11.33  1.45    Incubation time, h  Treatmenta  0  6  18  30  48  96  Control  319.1c  411.1c  563.4c  632.8  709.4  774.5c  RT1181  322.0c  424.3cd  564.3c  649.5  724.6  774.9c  RT1184  354.4d  445.2d  592.4d  657.2  739.8  782.9d  8184 Low  336.2cd  410.8c  55.89d  637.1  720.1  781.4d  8184 High  336.8cd  442.0cd  579.4cd  655.5  733.9  783.4d  SEMb  12.91  11.67  7.83  10.55  11.33  1.45  a Control = no enzyme added; RT1181 and RT1184 = enzymes added at 1.5 μL/g DM; 8184 Low = a mixture (1:1) of RT1181 and RT1184 added at 0.5 μL/g DM; 8184 High = a mixture (1:1) of RT1181 and RT1184 added at 1.5 μL/g DM. b SEM = standard error of the treatment means, n = 2. c,d Within columns, means without common superscripts differ (P < 0.05). View Large Table 9. Dry matter degradation (g/kg) of a mixture of alfalfa hay and corn silage, untreated or treated with enzyme products   Incubation time, h  Treatmenta  0  6  18  30  48  96  Control  319.1c  411.1c  563.4c  632.8  709.4  774.5c  RT1181  322.0c  424.3cd  564.3c  649.5  724.6  774.9c  RT1184  354.4d  445.2d  592.4d  657.2  739.8  782.9d  8184 Low  336.2cd  410.8c  55.89d  637.1  720.1  781.4d  8184 High  336.8cd  442.0cd  579.4cd  655.5  733.9  783.4d  SEMb  12.91  11.67  7.83  10.55  11.33  1.45    Incubation time, h  Treatmenta  0  6  18  30  48  96  Control  319.1c  411.1c  563.4c  632.8  709.4  774.5c  RT1181  322.0c  424.3cd  564.3c  649.5  724.6  774.9c  RT1184  354.4d  445.2d  592.4d  657.2  739.8  782.9d  8184 Low  336.2cd  410.8c  55.89d  637.1  720.1  781.4d  8184 High  336.8cd  442.0cd  579.4cd  655.5  733.9  783.4d  SEMb  12.91  11.67  7.83  10.55  11.33  1.45  a Control = no enzyme added; RT1181 and RT1184 = enzymes added at 1.5 μL/g DM; 8184 Low = a mixture (1:1) of RT1181 and RT1184 added at 0.5 μL/g DM; 8184 High = a mixture (1:1) of RT1181 and RT1184 added at 1.5 μL/g DM. b SEM = standard error of the treatment means, n = 2. c,d Within columns, means without common superscripts differ (P < 0.05). View Large Table 10. Fiber degradation (g/kg) of a mixture of alfalfa hay and corn silage, untreated or treated with enzyme products   Incubation time, h  Treatmenta  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  −36.8  21.0c  179.0cd  277.5  399.1  526.0c  RT1181  −27.6  32.8cd  164.6c  303.0  427.3  525.4c  RT1184  −13.5  50.9de  215.5d  317.9  444.6  536.6d  8184 Low  −7.1  26.4c  180.5cd  287.1  418.2  544.8e  8184 High  −14.0  59.6  207.9d  318.7  440.1  544.7e  SEMb  5.45  7.03  11.24  19.35  24.68  3.39  ——— ADF, g//kg ———  Control  −20.2  −10.2  119.2  225.1  354.7  487.9cd  RT1181  −21.0  10.2  111.4  258.1  380.3  480.5c  RT1184  −10.0  16.4  155.1  263.9  409.3  499.4cd  8184 Low  6.2  −19.6  118.4  234.5  370.5  504.1de  8184 High  −12.8  18.4  150.8  265.7  398.7  508.3e  SEM  7.49  15.14  15.45  16.22  24.81  5.56  ——— Hemicellulose, g/kg ———  Control  −61.8  67.7c  268.5cd  355.9  465.5  583.0c  RT1181  −37.4  66.5c  244.4c  370.2  497.6  592.6cd  RT1184  −18.9  102.6cd  305.1d  398.5  497.3  592.4cd  8184 Low  −27.2  95.4cd  273.4cd  365.7  489.6  605.7d  8184 High  −15.9  121.2d  293.3d  398.1  501.9  599.2cd  SEM  10.95  15.93  20.47  28.22  30.73  12.29    Incubation time, h  Treatmenta  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  −36.8  21.0c  179.0cd  277.5  399.1  526.0c  RT1181  −27.6  32.8cd  164.6c  303.0  427.3  525.4c  RT1184  −13.5  50.9de  215.5d  317.9  444.6  536.6d  8184 Low  −7.1  26.4c  180.5cd  287.1  418.2  544.8e  8184 High  −14.0  59.6  207.9d  318.7  440.1  544.7e  SEMb  5.45  7.03  11.24  19.35  24.68  3.39  ——— ADF, g//kg ———  Control  −20.2  −10.2  119.2  225.1  354.7  487.9cd  RT1181  −21.0  10.2  111.4  258.1  380.3  480.5c  RT1184  −10.0  16.4  155.1  263.9  409.3  499.4cd  8184 Low  6.2  −19.6  118.4  234.5  370.5  504.1de  8184 High  −12.8  18.4  150.8  265.7  398.7  508.3e  SEM  7.49  15.14  15.45  16.22  24.81  5.56  ——— Hemicellulose, g/kg ———  Control  −61.8  67.7c  268.5cd  355.9  465.5  583.0c  RT1181  −37.4  66.5c  244.4c  370.2  497.6  592.6cd  RT1184  −18.9  102.6cd  305.1d  398.5  497.3  592.4cd  8184 Low  −27.2  95.4cd  273.4cd  365.7  489.6  605.7d  8184 High  −15.9  121.2d  293.3d  398.1  501.9  599.2cd  SEM  10.95  15.93  20.47  28.22  30.73  12.29  a Control = no enzyme added; RT1181 and RT1184 = enzymes added at 1.5 μL/g DM; 8184 Low = a mixture (1:1) of RT1181 and RT1184 added at 0.5 μL/g DM; 8184 High = a mixture (1:1) of RT1181 and RT1184 added at 1.5 μL/g DM. b SEM = standard error of the treatment means, n = 2. c,d,e Within fractions and columns, means without common superscripts differ (P < 0.05). View Large Table 10. Fiber degradation (g/kg) of a mixture of alfalfa hay and corn silage, untreated or treated with enzyme products   Incubation time, h  Treatmenta  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  −36.8  21.0c  179.0cd  277.5  399.1  526.0c  RT1181  −27.6  32.8cd  164.6c  303.0  427.3  525.4c  RT1184  −13.5  50.9de  215.5d  317.9  444.6  536.6d  8184 Low  −7.1  26.4c  180.5cd  287.1  418.2  544.8e  8184 High  −14.0  59.6  207.9d  318.7  440.1  544.7e  SEMb  5.45  7.03  11.24  19.35  24.68  3.39  ——— ADF, g//kg ———  Control  −20.2  −10.2  119.2  225.1  354.7  487.9cd  RT1181  −21.0  10.2  111.4  258.1  380.3  480.5c  RT1184  −10.0  16.4  155.1  263.9  409.3  499.4cd  8184 Low  6.2  −19.6  118.4  234.5  370.5  504.1de  8184 High  −12.8  18.4  150.8  265.7  398.7  508.3e  SEM  7.49  15.14  15.45  16.22  24.81  5.56  ——— Hemicellulose, g/kg ———  Control  −61.8  67.7c  268.5cd  355.9  465.5  583.0c  RT1181  −37.4  66.5c  244.4c  370.2  497.6  592.6cd  RT1184  −18.9  102.6cd  305.1d  398.5  497.3  592.4cd  8184 Low  −27.2  95.4cd  273.4cd  365.7  489.6  605.7d  8184 High  −15.9  121.2d  293.3d  398.1  501.9  599.2cd  SEM  10.95  15.93  20.47  28.22  30.73  12.29    Incubation time, h  Treatmenta  0  6  18  30  48  96  ——— NDF, g/kg ———  Control  −36.8  21.0c  179.0cd  277.5  399.1  526.0c  RT1181  −27.6  32.8cd  164.6c  303.0  427.3  525.4c  RT1184  −13.5  50.9de  215.5d  317.9  444.6  536.6d  8184 Low  −7.1  26.4c  180.5cd  287.1  418.2  544.8e  8184 High  −14.0  59.6  207.9d  318.7  440.1  544.7e  SEMb  5.45  7.03  11.24  19.35  24.68  3.39  ——— ADF, g//kg ———  Control  −20.2  −10.2  119.2  225.1  354.7  487.9cd  RT1181  −21.0  10.2  111.4  258.1  380.3  480.5c  RT1184  −10.0  16.4  155.1  263.9  409.3  499.4cd  8184 Low  6.2  −19.6  118.4  234.5  370.5  504.1de  8184 High  −12.8  18.4  150.8  265.7  398.7  508.3e  SEM  7.49  15.14  15.45  16.22  24.81  5.56  ——— Hemicellulose, g/kg ———  Control  −61.8  67.7c  268.5cd  355.9  465.5  583.0c  RT1181  −37.4  66.5c  244.4c  370.2  497.6  592.6cd  RT1184  −18.9  102.6cd  305.1d  398.5  497.3  592.4cd  8184 Low  −27.2  95.4cd  273.4cd  365.7  489.6  605.7d  8184 High  −15.9  121.2d  293.3d  398.1  501.9  599.2cd  SEM  10.95  15.93  20.47  28.22  30.73  12.29  a Control = no enzyme added; RT1181 and RT1184 = enzymes added at 1.5 μL/g DM; 8184 Low = a mixture (1:1) of RT1181 and RT1184 added at 0.5 μL/g DM; 8184 High = a mixture (1:1) of RT1181 and RT1184 added at 1.5 μL/g DM. b SEM = standard error of the treatment means, n = 2. c,d,e Within fractions and columns, means without common superscripts differ (P < 0.05). View Large The combination of RT1181 and RT1184 showed values intermediate between the controls and RT1184 (Table 9), and treatment 8184 High tended (P < 0.10) to increase DMD at h 6 of incubation, accompanied by an increase (P < 0.05) in NDF and hemicellulose degradation. Because RT1181 failed to significantly increase DMD or fiber degradation in this experiment, it is reasonable to speculate that all increases found in the alfalfa-corn combination were due to the action of RT1184 alone. Furthermore, although our results were not entirely conclusive, it seems that the RT1184 application rate could be halved without losing effectiveness in fiber degradation, which agrees with preliminary data obtained by our lab (Colombatto and Beauchemin, unpublished data). Of particular interest was the fact that RT1184 and the two combinations of RT1181 and RT1184 increased (P < 0.05) both DMD and NDF end-point (96 h) degradation (Table 10). This is in contrast with what is generally observed when enzymes are added to forage (Yang et al., 1999; Colombatto, 2000). Although the increases in DMD are unlikely to be of biological significance, the extent of the improvement achieved with NDF degradation (+2.0, +3.5, and +3.5% for RT1184, 8184 Low, and 8184 High, respectively) is encouraging, especially when the treatments including RT1184 and 8184 High showed higher NDF degradation values at almost all incubation times. When the degradation of the nonfiber fraction was considered, it was found that the increases observed with preparation RT1184 during the first 18 h of incubation could not be only attributed to an increase in the fiber fraction, as the latter fraction explained between 25 and 50% of the increase in DMD. These findings are in complete agreement with those reported in Exp. 2 and indicate that this preparation acts mainly on nonfiber fractions. Furthermore, it was confirmed that this preparation was effective on forage mixtures as well as on pure alfalfa. Further work should focus on the complete characterization of this enzyme product, in terms of types and molecular properties of enzymes present, enzyme-feed specificity, and the effect of various application rates on the degradation of DM and fiber. Once these studies are complete, in vivo trials will be required to confirm the in vitro results presented here. Implications Results from this study suggest that it may not be possible to accurately predict the performance of a given enzyme additive based only on its biochemical properties and hydrolytic capacity in the absence of ruminal fluid. An additional in vitro step using ruminal fluid must be included before final selection of products for use in vivo is made. The two enzyme products selected here acted differently both in terms of feed specificity and whether the enzymic action started before or after feed ingestion. 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TI - Screening of exogenous enzymes for ruminant diets: Relationship between biochemical characteristics and in vitro ruminal degradation
JF - Journal of Animal Science
DO - 10.2527/2003.81102628x
DA - 2003-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/screening-of-exogenous-enzymes-for-ruminant-diets-relationship-between-Ls01Xolst9
SP - 2628
EP - 2638
VL - 81
IS - 10
DP - DeepDyve
ER -