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J. Anim. Sci. 2003. 81:2617-2627
© 2003 American Society of Animal Science

Effects of enzyme supplementation of a total mixed ration on microbial fermentation in continuous culture, maintained at high and low pH1

D. Colombatto*,2, G. Hervás{dagger}, W. Z. Yang* and K. A. Beauchemin*,3

* Agriculture and Agri-Food Canada, Lethbridge, AB, Canada, T1J 4B1 and and {dagger} Estación Agrícola Experimental (CSIC), Apdo. 788, 24080, León, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
A dual-flow continuous culture system was used to investigate the effects of pH and addition of an enzyme mixture to a total mixed ration (TMR) on fermentation, nutrient digestion, and microbial protein synthesis. A 4 x 4 Latin square design with a factorial arrangement of treatments was used, with four 9-d periods consisting of 6 d for adaptation and 3 d for measurements. Treatments were as follows: 1) high pH with control TMR, 2) high pH with TMR treated with enzyme, 3) low pH with control TMR, and 4) low pH with TMR treated with enzyme. Ranges of pH were 6.0 to 6.6 and 5.4 to 6.0 for high and low, respectively. Fermenters were fed twice daily a TMR consisting of 30% alfalfa hay, 30% corn silage, and 40% rolled corn (DM basis). The silage was milled fresh and the TMR was fed to the fermenters in fresh form (64% DM). The enzyme mixture was a commercial product of almost exclusive protease activity; it was applied daily to the fresh TMR and stored at 4°C for at least 12 h before feeding. Degradability of OM, NDF, ADF, and cellulose was decreased (P < 0.05) by low pH. Hemicellulose and protein degradation were not affected by pH. Enzyme addition increased (P < 0.01) NDF degradability (by 43% and 25% at high and low pH, respectively), largely as a result of an increase in hemicellulose degradation (by 79% and 51% at high and low pH, respectively). This improvement was supported by an increase (P < 0.05) in the xylanase and cellulase activities in the liquid phase of the fermenter contents. Total VFA were decreased (P < 0.05) by low pH, but were not affected by enzyme addition. Total bacterial numbers were increased (P < 0.03) at low pH and tended (P < 0.13) to increase with enzyme addition. Cellulolytic bacteria in effluent fluid were decreased (P < 0.02) at low pH but were unaffected by enzyme addition. Despite a large increase (P < 0.001) in protease activity, protein degradation was only numerically increased by enzyme addition. Microbial protein synthesis was higher (P < 0.10) at high pH but was not affected by enzyme addition. Methane production, expressed as a proportion of total gases, was decreased (P < 0.001) at low pH but was not affected by enzyme addition. It is concluded that it is possible to adapt the continuous culture system to use fresh feeds instead of dried feeds. Overall, the results indicate that the enzyme product used in this study has a potential to increase fiber degradability without increasing methane production.

Key Words: Digestion • Enzymes • pH • Rumen Fermentation


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Previous research has shown that the addition of enzyme mixtures can improve nutrient digestion and animal performance, as reviewed by Beauchemin et al. (2003)Go. However, the precise mode of action that enzyme mixtures have on ruminant diets remains elusive, and evidence suggests that the mechanism of action could be multifactorial in origin (McAllister et al., 2001Go).

Because the exogenous enzymes commonly employed as additives show lower optimal pH than ruminal enzymes, it has been speculated that their addition might be of greater benefit when conditions are suboptimal for ruminal digestion (i.e., cattle fed on high concentrate diets; Morgavi et al., 2000Go). Yang et al. (2002)Go used a dual-flow continuous culture system to evaluate the effects of pH and enzyme addition and concluded that, although the enzyme used in the study improved fiber digestion, its addition did not fully alleviate the depression in digestion brought about by the low pH. Colombatto (2000)Go used another enzyme mixture in a batch culture system and found that the rate of fiber digestion from alfalfa stems was improved only within the pH range of 6.2 to 6.8, without detectable effects at lower pH values. However, all of those studies were conducted using dried feeds, which differ from the on-farm situation, where feeds are offered fresh. The moisture content of feeds can have important implications given the recent findings of Wang et al. (2002)Go, who reported that enzymes were more effective on dried, as compared to fresh, silage.

In the present study, the influence of adding a selected enzyme mixture to a total mixed ration (TMR) for dairy cattle, offered fresh, on the microbial fermentation in continuous culture was examined. In addition, two fermentation pH ranges (5.4 to 6.0 and 6.0 to 6.7) were maintained by adjusting the concentration of the artificial saliva. Our hypothesis was that the enzyme mixture would improve the degradability of the diets and that the extent of the improvement would be lower at low pH than at high pH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Diets and Enzyme Product
The TMR used in this study consisted of 30% alfalfa hay, 30% corn silage, and 40% rolled corn grain (DM basis). The alfalfa hay was ground to pass a 4.5-mm screen (Arthur H. Thomas Co., Philadelphia, PA), whereas the rolled corn was processed in a Knifetec 1095 sample mill (Foss Tecator, Höganäs, Sweden) for 2 s to achieve partial rupture of the grains. Both substrates were stored at room temperature until used. Corn silage was sampled from different sites within a bunker silo located at the Lethbridge Research Centre (Lethbridge, AB) and stored at -40°C until used. When required, a sample of the silage (enough for 3 d of feeding) was thawed and processed fresh in the Knifetec mill for 10 s. Processed samples were stored at 4°C for a maximum of 3 d. The TMR was prepared every 3 d in 1-L plastic containers by weighing the individual feed components. The contents were mixed thoroughly and stored at 4°C. Chemical composition of the individual components and of the TMR is shown in Table 1Go.


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  		Table 1. Chemical composition (g/kg of DM) of the feeds and of the total mixed ration (TMR)
 
The enzyme product was selected based on in vitro studies carried out previously (our unpublished observations) and was obtained from Cargill, Inc. (St. Louis, MO). It is a commercial product derived from Bacillus licheniformis, compliant with the current specifications for food-grade enzymes and generally recognized as safe. The product was evaluated for its enzyme activities at pH 6.0 and 39°C (reflective of ruminal conditions) and was found to contain negligible amounts of cellulase, hemicellulase, and {alpha}-amylase activities (Colombatto et al. 2002aGo). Protease activity in the enzyme product was determined at pH 6.0 and 39°C using 0.4% (wt/vol) azocasein as substrate (Bhat and Wood, 1989Go). Briefly, a reaction mixture containing 0.5 mL of azocasein, 0.5 mL of citrate-phosphate buffer, and 25 µL of enzyme (diluted 1:100 in distilled water) was incubated at 39°C for 15 min. The unhydrolyzed azocasein was precipitated by adding 80 µL of 25% (wt/vol) trichloroacetic acid and then removed by centrifugation (2,040 x g, 10 min, room temperature). A 0.5-mL supernatant sample was mixed with 0.5 mL of 0.5 M NaOH and the absorbance read at 420 nm against a reagent blank. Enzyme (no substrate) and substrate (no enzyme) blanks were also included for correction. One unit of protease activity was defined as the absorbance measured at 420 nm by the action of 10 µg of a standard protease ( Streptomyces griseus, Type XIV, Sigma Chemicals, St. Louis, MO) assayed under identical conditions. Based on these experimental conditions, the protease activity of the exogenous enzyme product was determined to be 4,507 units/mL (SD = 161.0, n = 5).

Apparatus and Experimental Design
A four-unit dual-flow continuous culture system similar to that of Hoover et al. (1989)Go was used in four consecutive periods. Ruminal fluid inoculum was obtained 2 h after feeding from three ruminally fistulated lactating dairy cows fed a diet similar to that provided to the fermenters. The donor cows were cared for in accordance with the guidelines established by the Canadian Council on Animal Care (1993)Go. Mixed ruminal contents were homogenized in a Waring blender (Waring Product Division, New Hartford, CT) for 1 min, under a stream of oxygen-free CO2. The homogenized material was then strained through four layers of cheesecloth and transferred to the laboratory in prewarmed thermally insultated flasks. Anaerobic conditions were maintained by infusion of CO2 at a rate of 15 mL/min. Artificial saliva (McDougall, 1948Go) was continuously infused into the fermenters. During each period, two fermenters received saliva at the normal concentration and the other two fermenters received saliva that was diluted in distilled water to obtain a concentration equivalent to 60% of the normal. The artificial saliva contained 0.2 g/L of urea to simulate recycled nitrogen and 0.015 g of ammonia 15N ([15NH4]2SO4, 10.6% atom percentage 15N; Isotec, Miamisburg, OH; daily amount of 15N provided into each fermenter was about 1.5 mg). Liquid and solids dilution rates were maintained constant (10 and 4.5 %/h, respectively). A total of 80 g/d of DM was fed in two equal meals at 0900 and 2100.

Treatments used in this study were as follows: high pH with control TMR (HC), high pH with TMR treated with enzymes (HT), low pH with control TMR (LC), and low pH with TMR treated with enzymes (LT). Treatments HC and HT received the "normal" saliva, whereas treatments LC and LT received the diluted (60% of the normal) saliva. For application of the enzyme, 60 µL of undiluted enzyme product was dissolved into 440 µL of distilled water and then added to 40 g (DM basis) TMR (stored in 250-mL plastic containers) using a pipette. The control treatments received 500 µL of distilled water. Upon enzyme addition, the TMR in the plastic containers was mixed by inversion several times. Enzyme-feed interaction time ranged between 12 and 24 h at 4°C.

The experimental design was a 4 x 4 Latin square with four 9-d periods. Each experimental period consisted of 6 d for adaptation and 3 d for sampling. During sampling days, collection vessels were maintained at 4°C to impede microbial action. Solid and liquid effluents were mixed, and a 250-mL sample was centrifuged at 16,000 x g for 40 min at 4°C to determine effluent DM; a second, 500-mL sample was centrifuged at 16,000 x g for 40 min at 4°C and sediments were dried at 55°C and then analyzed for ash, N, NDF, ADF, ADL, and starch. On d 1 and 2 of each sampling period, fermenter pH was measured every hour from 0800 to 2100 using a pH probe inserted into the fermenters. Fluid samples from the filtrate were obtained immediately before the feed provision in the morning, and then at 2, 5, 8, and 12 h after feed provision for ammonia and VFA determination. A 5-mL subsample of filtered fluid was acidified with 1 mL of 1% sulfuric acid (vol/vol) for ammonia determination, whereas another 5-mL subsample was acidified with 1 mL of 25% metaphosphoric acid (wt/vol) for VFA analysis. The samples were stored frozen at -40°C until analyzed. Six hours after the morning feed provision (i.e., 1500), gas samples were taken for compositional analysis (CO2 and CH4). At the same time, a 2.0-mL sample of ruminal fluid was taken from the fermenters for quantification of total and cellulolytic bacteria, and an additional, 1.5-mL sample was withdrawn for determination of enzyme activities.

Bacteria were isolated from fermenter flasks on the last day of each period. Fermenter contents were homogenized for 1 min with a Waring blender (Waring Products Division, New Hartford, CT) at slow speed to dislodge solid-phase bacteria and then strained through four layers of cheesecloth. The filtrate was centrifuged at 1,196 x g for 15 min and 4°C to remove particle-associated bacteria, feed particles, and protozoa and then at 16,000 x g for 40 min and 4°C to isolate the bacterial pellet. The pellets were lyophilized, further ground using a mortar and pestle, and then analyzed for 15N enrichment. Apparent and true (i.e., corrected for microbial portion) digestion of DM, OM, and N were calculated. Digestion of NDF, ADF, ADL, and starch were also determined.

Bacterial Counts
For the enumeration of the total viable bacteria population, anaerobic serial dilutions (10-6 to 10-9) of filtered fermenter contents were prepared as described by Bryant and Burkey (1953)Go using a medium containing 0.1% peptone, 0.1% resazurin, 0.05% cysteine, and 0.35% Na2CO3. Each dilution was inoculated in triplicate into separate roll tubes containing cellobiose, xylan, starch, and glucose (0.5 mg/mL each). Viable colonies were enumerated after 48 h of incubation at 39°C. Cellulolytic bacteria were enumerated following a 14-d incubation at 39°C in triplicate tubes with each of the dilutions (10-1 to 10-4), using filter paper (Whatman No. 1) as the sole carbohydrate source. The most probable number procedure described by Garthright (1998)Go was used. Prior to statistical analysis, microbial data were subjected to log transformation to normalize the distribution of the error (Dehority et al., 1989Go).

Determination of Enzyme Activities
Enzyme activities in the liquid phase were determined as described previously (Colombatto et al., 2003Go). Endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91), ß-D-glucosidase (EC 3.2.1.21), xylanase (EC 3.2.1.8), ß-D-xylosidase (EC 3.2.1.37), protease, and {alpha}-L-arabinofuranosidase (EC 3.2.1.55) activities were determined. Oat spelt xylan and medium-viscosity carboxymethylcellulose (Sigma Chemicals; 10 mg/mL) were used as substrates for xylanase and endoglucanase activity determination, respectively. Forty microliters of effluent fluid was incubated with 1 mL of substrate, 0.90 mL of buffer (0.1 M citrate-phosphate buffer, pH 6.0), and 0.06 mL of distilled water. Incubations were performed in triplicate for 60 min (xylanase) or 120 min (endoglucanase) at 39°C. The enzymatic reaction was terminated by adding dinitrosalicylic acid reagent, and absorbance was read at 530 nm using a MRX-HD plate reader (Dynatech Laboratories Inc., Chantilly, VA). The absorbance values were converted to reducing sugars using standard xylose or glucose curves, developed under identical conditions. Blanks, substrate alone (i.e., no enzyme) and enzyme alone (i.e., no substrate) were also included to correct for substrate autolysis and sugars present in the enzyme sample, respectively. One unit of activity was defined as the amount of enzyme required to release 1 nmol of xylose or glucose equivalent per minute, under the conditions of the assay. Protease activity was assayed at pH 6.8 using a 0.4% (wt/vol) solution of azocasein as described above, except that incubation time was 120 min, and 40 µL of sample was incubated. One unit of protease activity was defined as the absorbance measured at 420 nm by the action of 1 µg of a standard protease (Streptomyces griseus, Type XIV, Sigma Chemicals), assayed under identical conditions and simultaneously to each incubation series. We used 1 µg as a standard on this occasion because of the different assay lengths used. If 10 µg were used, the absorbance would have been too high to fall within the linear range of optical density.

For aryl-glycosidase activity determinations, stock solutions (1 mM) of p-nitrophenyl (p-NP) derivatives were used. Substrates, obtained from Sigma Chemicals, were p-NP-ß-D-cellobioside, p-NP-ß-D-glucopyranoside, p-NP-ß-D-xylopyranoside, and p-NP-{alpha}-L-arabinofuranoside. Undiluted enzyme samples (20 µL) were incubated with 80 µL of corresponding substrate (prepared in buffer, pH 6.0) at 39°C for 180 min. The reaction was terminated by the addition of 1 vol of glycine-NaOH buffer (0.4 M, pH 10.8). The release of p-nitrophenol was determined colorimetrically at 420 nm. One unit of enzyme activity was defined as the amount of enzyme required to release 1 nmol p-nitrophenol per minute, under the conditions of the assay.

Chemical Analysis
Effluent DM was calculated by drying at 55°C in a forced-air oven for 48 h. The DM content of diets and bacterial samples were determined by drying at 110°C for 24 h. Organic matter was determined by difference following ashing at 500°C overnight. Crude protein content (N x 6.25) of samples was determined by flash combustion, chromatographic separation, and thermal conductivity (Carlo Erba Instruments, Milan, Italy), following the procedures outlined by AOAC (1990)Go. The methods of Van Soest et al. (1991)Go were used to determine neutral and acid detergent fiber using the ANKOM200 (ANKOM Corp., Fairport, NY) fiber analyzer. Heat-stable amylase was used during the NDF procedure, but sodium sulfite was omitted. Starch was determined by enzymatic hydrolysis of {alpha}-linked glucose polymers as described by Rode et al. (1999)Go.

Ammonia content was determined using a modification of the Berthelot reaction (Verdouw, 1978Go). The VFA were separated and quantified by gas chromatography (GC, Hewlett Packard 5890, Agilent Technologies, Mississauga, ON) using a 30-m (0.32-mm i.d.) fused-silica column (Nukol column, Sigma-Aldrich Canada Ltd., Oakville, ON). Lactic acid contents at 2 h postfeeding were determined by derivatization with boron trifluoride-methanol (14% BF3 in methanol) following the procedures described in a Supelco bulletin (Supelco, 1998Go). The resultant methyl esters were analyzed by GC using helium as a carrier (28 cm/s). A sample of methyl DL-lactate was run to confirm the retention time of the derivative.

Headspace samples of gas were removed 6 h after feeding via the port (with an inserted GC septum) into a 10-mL syringe fitted with a 26-gauge needle (Leurlock). The sample was immediately injected into an evacuated 1-dram vial, and analyzed by GC (Micro GC CP3900, Varian Specialties Ltd., Brockville, ON, Canada), using a 10-m PoraPlot Q column.

Enrichment of 15N in the bacterial pellets isolated from the fermenter contents was determined by flash combustion (Model 1500, Carlo Erba Instruments, Milan, Italy) with isotope ratio mass spectrometry (VG Isotech, Middlewich, UK). A correction for natural abundance of 15N-enriched bacteria was made by running an additional experimental period without infusion of 15N using two fermenters at high pH and two at low pH. Bacterial production was estimated by the ratio of 15N flow in the effluent to 15N enrichment of the bacterial pellet.

Statistical Analysis
Data were analyzed using the Mixed procedures of SAS (SAS Inst. Inc., Cary, NC), using a model that included pH, enzyme, and their interaction as fixed effects. Fermenter and period were considered random effects. Differences among means were declared significant at P < 0.05, whereas trends were discussed at P < 0.15, unless stated otherwise.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
The TMR used in the present study (Table 1Go) contained a forage:concentrate ratio of 60:40 and a CP content of 14.3% (or 14.5% when considering the inclusion of urea and 15N), which is typical of diets fed commercially to dairy cows in mid to later lactation. This higher forage diet was used in order to evaluate the effects of the enzyme on fiber digestion. In addition, the corn silage used here was ground fresh without generation of heat, and the particle size distribution obtained, as estimated using wet-sieving techniques, was similar to that of the ruminal contents. The sieves used were of 9.5, 4.75, 3.35, 1.18, 0.6, 0.15, and 0 mm, and retained 0.1, 1.3, 1.6, 11.7, 6.1, 24.2, and 55.0% of the corn silage after milling, respectively. The ranges of pH obtained by altering the saliva concentration are shown in Figure 1Go. Clearly, two different pH profiles were obtained as planned. Enzyme addition or its interaction with treatment did not affect (P > 0.05) pH at any time.



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  		Figure 1. Diurnal fluctuation of pH in continuous culture fermenters after feed addition (0900) as affected by enzyme supplementation. Values are least squares means, and vertical bars indicate the SEM of the pH x enzyme interaction (n = 4).

 
True OM degradation was lower (P < 0.05) at low pH (Table 2Go); however, true DM degradation only tended (P < 0.07) to be lower. Enzyme addition did not affect (P > 0.20) true DM or OM degradation. Fiber (NDF and ADF) degradation was greatly decreased (P < 0.001) at low pH, whereas enzyme addition increased (P < 0.004) NDF degradation. Hemicellulose degradation was increased (P < 0.001) as a result of enzyme addition, but cellulose degradation was unaffected (P > 0.32). Both true degradation of CP and starch degradation were unaffected (P > 0.15) by the treatments.


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  		Table 2. Effects of pH and enzyme addition on DM, OM, fiber and starch degradation in continuous culture
 
Total VFA concentration was lower (P < 0.007) at low pH (Table 3Go). The branched-chain VFA concentration also decreased with low pH (P < 0.001). The proportions of the VFA varied according to the particular VFA under consideration. Maintaining the fermenters at high pH increased (P < 0.01) the molar proportions of acetate, butyrate, isobutyrate, and isovalerate, with caproate showing a trend (P < 0.14) toward an increase. However, high pH decreased (P < 0.01) the proportions of propionate and valerate. The acetate:propionate ratio was lower (P < 0.001) at low pH than at high pH. There was no effect (P > 0.20) of enzyme addition on the concentration or molar proportion of any VFA. The concentrations of lactic acid were low and probably not biologically meaningful; however, a trend (P < 0.10) toward higher concentrations at the high pH was observed (Table 3Go).


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  		Table 3. Effects of pH and enzyme addition on VFA, lactic acid, and gas concentrations in continuous culturea
 
The contribution of methane to the total gas composition was greatly decreased (P < 0.001) by low pH (Table 3Go), whereas the CO2 proportion was higher (P < 0.04) at high pH. Total bacterial counts were higher (P < 0.03) at low pH than at high pH (Table 4Go); however, the cellulolytic bacteria numbers were higher (P < 0.01) at high pH. Enzyme addition tended (P < 0.13) to increase the total viable bacterial count, but had no effect (P > 0.88) on the cellulolytic bacteria populations.


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  		Table 4. Effects of pH and enzyme addition on total viable and cellulolytic bacteria counts in continuous culture at 6 h after provision of feed to the fermentersa
 
The enzymic activities measured for the liquid phase of fermenter contents are shown in Table 5Go. Endoglucanase and ß-D-xylosidase activities were lower (P < 0.04) at low pH, whereas exoglucanase activity tended (P < 0.10) to be decreased. In contrast, protease activity was higher (P < 0.001) at lower pH, largely due to the increase in activity shown in treatment LT. Addition of exogenous enzyme increased (P < 0.02) xylanase, endoglucanase, and protease activities, and tended to increase ß-D-glucosidase (P < 0.06) and exoglucanase (P < 0.11). A significant (P < 0.01) pH x enzyme interaction was detected for ß-D-xylosidase, as exogenous enzyme addition appeared to increase this activity at high pH, but decrease it at low pH. For protease activity, the significant pH x enzyme interaction (P < 0.001) was due to the large increase in activity shown by LT, as mentioned. Only {alpha}-L-arabinofuranosidase remained unaltered (P > 0.15) by pH or enzyme treatments.


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  		Table 5. Effects of pH and enzyme addition on enzymic activities at 6 h after feeding.
 
Data on N metabolism are presented in Table 6Go. Total N flow tended (P < 0.14) to be greater at high pH, whereas exogenous enzyme addition tended (P < 0.07) to decrease it. Neither bacterial nor dietary N flow was affected by treatments (P > 0.15). The ammonia concentrations determined were extremely low and were greater at high pH (P < 0.002), with a trend (P < 0.06) to be increased by the exogenous enzyme addition. As a result, the efficiency of microbial protein synthesis was greater (P < 0.09) at high pH than at low pH.


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  		Table 6. Effect of pH and enzymes on the N metabolism of ruminal microbes in continuous culture
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
The experimental approach used in the present study differs from others carried out in the past. First, the pH was adjusted to yield different pH ranges by varying the concentration of the artificial saliva, but without affecting its composition or the total volumes infused daily. This was done in order to simulate the reductions in salivation that typically occur when cattle are fed high concentrate diets (Van Soest, 1994Go). Other workers have altered pH by changing the saliva composition (Slyter et al., 1966Go; Yang et al., 2002Go), or adding concentrated amounts of acids or bases in an effort to keep pH constant (de Veth and Kolver, 2001Go; Calsamiglia et al., 2002Go). However, as discussed by de Veth and Kolver (2001)Go, "inorganic acids or bases may not fully represent the organic acids and bases that regulate ruminal pH". In addition, it is unknown whether the abrupt changes in pH brought about by the addition of acids (e.g., from 6.3 to 5.4 in 5 min) have a direct effect on the microbial populations. A valid counterargument is that when the main objective is to study the effect of pH on microbial fermentation this factor should be isolated and kept as constant as possible. However, a constant ruminal pH is not biologically meaningful; therefore, we feel that alteration of the range of pH at which the rumen normally works as a function of diet is more appropriate. As a result, two pH profiles were obtained, which influenced the fermentation patterns and changed the responses to enzyme addition.

The other modification used in the present study was the use of fresh forages instead of oven- or freeze-dried. This is relevant to the technical aspect of the in vitro techniques currently employed for feed evaluation, as it helps us bridge the gap between in vitro and in vivo studies (Bowman et al., 2002Go). In addition, it has been shown that exogenous enzymes appear to work better in dried than in fresh corn silage (Wang et al., 2002Go), which might help explain why certain enzyme products are effective in typical in vitro assays but not when added in vivo. The use of fresh feeds in future in vitro studies with enzyme products will be advantageous.

Lowering the pH range in the fermenters resulted in a decrease in OM and fiber degradation, which is consistent with earlier in vivo (Mould and Ørskov, 1983Go) and continuous culture results (Erfle et al., 1982Go; Calsamiglia et al., 2002Go). The decrease in fiber degradation seen in this study most likely resulted from the reduction in the numbers of cellulolytic bacteria observed at these low pH values, which agrees with previous reports (Stewart, 1977Go; Russell and Dombrowsky, 1980). Addition of an enzyme mixture greatly increased NDF degradation, mostly at the higher pH range. These findings support our hypothesis and concur with a recent study of Yang et al. (2002)Go, who reported a larger (17.6 vs 7.6%) increase in NDF degradation due to enzymes when pH was maintained above 6.0 vs. below 6.0. In that study, the results were somewhat unexpected given that the enzyme product showed optimal fibrolytic activities under acidic conditions. Our findings, together with Yang et al. (2002)Go, refute the hypothesis that enzymes are of greater benefit when pH conditions are suboptimal (Morgavi et al., 2000Go; Colombatto et al., 2002bGo). The enzyme product used here has been shown to work optimally at high (> 8.0) pH conditions (our unpublished observations) but does not contain detectable amounts of fibrolytic activities (Colombatto et al., 2002aGo). Indeed, it is a concentrated protease product. It is therefore unlikely that the enzyme per se increased fiber degradation, rather it seems that the enzyme exerted an indirect effect. One possibility is that the enzyme product acted by removing structural barriers that allowed faster microbial access to degradable fiber in the alfalfa, a hypothesis also suggested by Nsereko et al. (2000)Go, who used a different enzyme product. This faster microbial access to degradable fiber is supported by the observed increase in most of the enzymic activities in the ruminal fluid when enzymes were added. Because the pretreatment period (12 to 24 h) was carried out at 4°C, enzymic activity on the feed before being added to the fermenters would have been minimized. Even when it is possible that the length of the pretreatment period might have partly offset the inhibitory effect of the low temperature, it seems more likely that the effects were exerted during the first stages of the fermentation. More research is required to examine the ideal pretreatment conditions for this particular enzyme product.

When the digestibility of the fibrous fractions was examined, hemicellulose digestion was not affected by low pH, possibly indicating that the hemicellulolytic bacteria remained functional. This idea is reinforced by the fact that xylanase activity was not affected by pH. Russell and Dombrowski (1980)Go reported that the hemicellulolytic species Prevotella ruminicola is more resistant to low pH than cellulolytic bacteria. Unfortunately, most of the previous experiments examining the effect of pH on fiber degradability have not reported the degradation of the hemicellulose fraction alone, thereby preventing direct comparisons. Recalculating original data from Yang et al. (2002)Go, hemicellulose degradation was affected only when pH was kept at 5.5, but not when it was kept at 6.0 or 6.5. However, Yang et al. (2002)Go reported that ADF degradation was linearly decreased with decreasing pH. These findings may suggest that the effects of pH on fiber degradability will be influenced by the fiber composition of the feed under study. Thus, the negative effects of pH on fiber digestion may be larger as cellulose content in the substrate increases.

Despite a numerical increase in cellulose degradation for HT compared to HC, most of the increase in NDF degradation for the HT and LT treatments seems to have been caused by an increase in hemicellulose degradation. This is consistent with Dong et al. (1999)Go and with previous data obtained in our lab using alfalfa hay or alfalfa-corn silage mixtures treated with the same enzyme product (unpublished data). The numerical increase in cellulose degradation observed for HT probably resulted from the more extensive action of the exogenous enzyme at these pH conditions, which may have allowed cellulolytic microbes further access to their substrate. Overall, these findings are supported by the observed increases in xylanase and cellulase activities. However, these findings must be qualified against the low ammonia concentrations observed (Table 6Go), because it is possible that had the ammonia concentrations been higher, results could have differed. Recently, Griswold et al. (2003)Go observed that hemicellulose degradation was not affected by low ammonia concentrations, whereas the ADF degradation was decreased at those low concentrations. In our study, addition of the enzyme mixture might have provided additional nitrogenous growth factors that were rapidly used by the hemicellulolytic bacteria to grow and degrade their substrate.

The degradation of starch was not affected by treatment but showed high values (>90%) in all cases. It is likely that this high degradation partly explained the relatively low fiber degradation (<35%) achieved. High starch degradation usually leads to a rapid release of VFA, which lowers the pH, thereby affecting fiber degradation (Mould and Ørskov, 1983Go). Mansfield et al. (1995)Go suggested that this phenomenon was probably linked to the amount of concentrate supplied to the fermenters, implying a shift from fibrous to nonfibrous carbohydrate fermentation.

The VFA profiles observed in the present study are in general agreement with previous reports (Erfle et al., 1982Go; Calsamiglia et al., 2002Go). However, the acetate:propionate ratio of all treatments in our study was considerably lower than that published in those reports but very similar to that reported by Yang et al. (2002)Go, who used the same continuous culture system used here. The increase in BCVFA concentration at higher pH conditions probably supported the higher growth of cellulolytic bacteria observed. On that note, it is important to point out that because the cellulolytic numbers were obtained from the liquid phase of the fermenters, the absolute values reported here are likely to be a significant underestimate. It is well known that most of the cellulolytic microbial biomass is particle-associated (Craig et al., 1987Go). Addition of the exogenous enzyme product did not affect the VFA profiles, which agrees with previous in vivo (Feng et al., 1996Go; Yang et al., 1999) and in vitro (Yang et al., 2002Go; Colombatto et al., 2003Go) studies using a range of products and diets. The amounts of lactic acid observed at 2 h postfeeding were low, which is consistent with a well-adapted ruminal population and with the type of diet used. Slyter et al. (1966)Go did not detect any lactic acid when alfalfa hay was fed in continuous culture, even at pH 5.0.

Crude protein degradation was not affected by the low pH conditions, which is in contrast to previous reports (Erfle et al., 1982Go; Calsamiglia et al., 2002Go) but which agrees with Yang et al. (2002)Go. Probable reasons for this discrepancy include differences in the diets used in those studies and the characteristics of the ruminal fluids used as initial inocula for the fermenters. In all four experimental periods of our study, the pH of the ruminal fluid used to inoculate the fermenters was around 5.5, suggesting that the cows had established populations working under those acidic conditions. According to Wallace et al. (1997)Go, proteolytic activity in the rumen has a broad optimum pH (5.5 to 7.0), and amylolytic bacteria (more likely to be found at acidic conditions) tend to have more proteolytic activity than their cellulolytic counterparts. Therefore, it is likely that the amylolytic populations contributed to the protein degradation at low pH. Interestingly, CP degradability was only numerically increased by enzyme addition (average of 6.7%), despite a large increase in ruminal protease activity (almost fourfold at high pH, and almost ninefold at low pH) in the enzyme-added treatments. Despite the lack of statistical significance, this small increase may have biological implications, as it has been proposed that structural proteins serve a cross-linking role in legume cell walls (Fry, 1986Go; Jung, 1997Go). It is speculated that the exogenous enzyme might have cleaved specific linkages between protein and fiber in the cell wall, thereby increasing the rate of fiber degradation by ruminal populations. Further research on the type of proteases present in this enzyme product should shed more light on this issue.

The total flow of N to the duodenum showed a trend toward reduction with low pH or with enzyme addition. However, neither the bacterial nor the dietary N flows were affected. Yang et al. (2002)Go also found no effect of enzymes or pH on N metabolism in continuous culture. The ammonia concentrations found in this study were extremely low, which is difficult to explain given that protein degradation and branched-chain VFA production (which are derived from protein and amino acid degradation) were within normal ranges. It is unlikely that ammonia was in short supply, as the inclusion of urea (0.2 g/L) and 15N in the buffer, plus the dietary degradable protein should have ensured an adequate ammonia provision. Griswold et al. (2003)Go reported values similar to those reported here but only when urea was omitted from the buffer. Those authors also suggested that ammonia provision was not crucial for the hemicellulose-degrading bacteria to effectively degrade the hemicellulose. In spite of this, relative differences in ammonia concentrations were observed, as ammonia concentrations were lower with lower pH, and tended to increase with enzyme addition. The decrease in ammonia concentration with low ruminal pH agrees with previous studies (Erfle et al., 1982Go; Lana et al., 1998Go). A trend toward higher ammonia concentrations with enzyme addition might have resulted from the numerical increases in protein degradation observed. The efficiency of microbial protein synthesis increased at high pH values, which is in agreement with Hoover and Miller-Webster (1992)Go, who reported that efficiency of microbial protein synthesis in vitro was negatively affected when pH dropped below 5.5.

Total microbial counts increased at low pH values, in agreement with Slyter et al. (1966)Go and Erfle et al. (1982)Go. Although several ruminal bacteria have been shown to wash out at pH lower than 6.0 (Russell and Dombrowsky, 1980), it is likely that the loss of these species allowed an enhanced growth of species more tolerant to low pH conditions (Erfle et al., 1982Go). However, the shift in microbial populations evidenced by changes in fiber degradation and VFA profiles did not alter microbial growth, as indicated by the lack of changes in bacterial N synthesis. Enzyme addition tended to increase the total microbial numbers, in partial agreement with recent reports (Wang et al., 2001Go; Nsereko et al., 2002Go). In the work of Nsereko et al. (2002)Go, it was found that a fibrolytic enzyme product increased the numbers of bacteria that utilize hemicellulose or secondary products of cellulose digestion, but did not affect the numbers of cellulolytic bacteria. The higher xylanase activity found in the present study and by Wang et al. (2001)Go seem to support this idea; however, the use of enzyme products differing in their biochemical properties make direct comparisons difficult.

Although it was not a prime objective of this work, methane production was also determined. Lowering the pH range greatly decreased the contribution of methane to the total fermentation gases, which is in agreement with previous studies (Erfle et al., 1982Go; Van Kessel and Russell, 1996Go; Lana et al., 1998Go). Moreover, the decrease in methane was obtained despite using the same diet in all treatments, suggesting that pH was the leading factor for this decrease (Russell, 1998Go). Interestingly, enzyme addition did not affect methane production, which is in contrast with Dong et al. (1999)Go, who found an increase in methane production as a result of addition of exogenous enzyme. If we consider that the large increases in NDF degradability (43% and 26% at high and low pH, respectively) were achieved without increasing the methane production, it follows that this enzyme product has significant potential to be included as a feed additive for ruminants.

In conclusion, the addition of a novel proteolytic enzyme product to a TMR evaluated using continuous culture enhanced fiber (especially hemicellulose) degradation, most likely owing to the removal of structural barriers that allowed faster microbial access to the degradable substrates. Our hypothesis that the effects of this enzyme would be larger at higher pH was confirmed. More research is required to investigate the characteristics of this enzyme product that likely cause these responses.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
The present research has demonstrated that the dual-flow continuous culture system can be adapted to use fresh instead of oven- or freeze-dried feeds. Also, the manipulation of pH by altering the artificial saliva concentration allowed two pH profiles to be used. Addition of an exogenous proteolytic enzyme under different pH conditions enhanced fiber degradation with only a numerical increase in protein degradation. Overall, these findings further suggest that the mode of action of exogenous enzymes in ruminants is a combination of direct and indirect effects, which are exerted both over the feeds and the microbial populations in the rumen. In vivo research should be conducted before this enzyme product can be commercialized as a feed additive for ruminants.


   Footnotes
 
1 Lethbridge Research Centre contribution No. 387 03003. We thank A. F. Furtado, D. D. Vedres, and G. R. Bowman for skilled technical assistance; K. Koenig for helpful suggestions; and T. Entz for help with statistical analysis. Hervás acknowledges receipt of a fellowship from the Spanish Ministry of Science and Technology (MCyT). Back

2 Present address: Departamento de Producción Animal, Facultad de Agronomia, Universidad de Buenos Aires, Argentina. E-mail: colombat{at}agro.uba.ar. Back

3 Correspondence: Box 3000 (phone: 403-317-2235; fax: 403-317-2182; E-mail: beauchemin{at}agr.gc.ca).

Received for publication January 30, 2003. Accepted for publication July 4, 2003.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


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