| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Agriculture and Agri-Food Canada, Lethbridge, AB, Canada, T1J 4B1 and
and
Institute National de la Recherche Agronomique, Centre Clermont-Theix, 63122 Saint-Genès-Champanelle, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterial and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
Key Words: Degradation • Enzymes • Fiber • Ruminants • Screening
|
Introduction |
|---|
|
TopAbstractIntroductionMaterial and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
; 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 |
|---|
|
TopAbstractIntroductionMaterial and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
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), ß-1,3-ß-1,4-glucanase (EC 3.2.1.73), ß-1,3-glucanase (EC 3.2.1.6), and 
-amylase (3.2.1.1), respectively. In addition, barley ß-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 ß-D-glucosidase (EC 3.2.1.21), ß-D-xylosidase (EC 3.2.1.37), -L-arabinofuranosidase (EC 3.2.1.55), ß-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, -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 |
|---|
|
TopAbstractIntroductionMaterial and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
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.
|
). 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 ß-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 
-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).
|
). 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).
|
). 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 ß-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.
|
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, -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 -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.
|
(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.
|
|
). 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).
|
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).
|
|
), 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 |
|---|
|
TopAbstractIntroductionMaterial and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
|
Footnotes |
|---|
2 Present address: Departamento de Producción Animal, Facultad de Agronomía, Universidad de Buenos Aires, Argentina. E-mail: colombat{at}agro.uba.ar.
3 Correspondence: Box 3000 (phone: 1-403-317-2235; fax: 1-403-317-2182; e-mail: beauchemin{at}agr.gc.ca).
Received for publication January 30, 2003. Accepted for publication June 5, 2003.
|
Literature Cited |
|---|
|
TopAbstractIntroductionMaterial and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
-amylase and protease activity is related to the virulence of Aspergillus flavus isolates. J. Food Prot. 64:401–404.[Medline]
-L-Arabinofuranosidase from Ruminococcus albus 8: Purification and possible role in hydrolysis of alfalfa cell wall. Appl. Environ. Microbiol. 47:1135–1140.This article has been cited by other articles:
|
|
 |
|
  J.-S. Eun and K. A. Beauchemin Enhancing In Vitro Degradation of Alfalfa Hay and Corn Silage Using Feed Enzymes J Dairy Sci, June 1, 2007; 90(6): 2839 - 2851. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-S. Eun, K. A. Beauchemin, and H. Schulze Use of Exogenous Fibrolytic Enzymes to Enhance In Vitro Fermentation of Alfalfa Hay and Corn Silage J Dairy Sci, March 1, 2007; 90(3): 1440 - 1451. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-S. Eun and K. A. Beauchemin Effects of a Proteolytic Feed Enzyme on Intake, Digestion, Ruminal Fermentation, and Milk Production J Dairy Sci, June 1, 2005; 88(6): 2140 - 2153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. McGinn, K. A. Beauchemin, T. Coates, and D. Colombatto Methane emissions from beef cattle: Effects of monensin, sunflower oil, enzymes, yeast, and fumaric acid J Anim Sci, November 1, 2004; 82(11): 3346 - 3356. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
