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ANIMAL NUTRITION |
* Agriculture and Agri-Food Canada Research Centre, Lethbridge, Alberta, T1J 4B1 Canada and
and
Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan 84322-4815
Abstract
The effects of pretreating wheat straw with alkali on the efficacy of exogenous fibrolytic enzymes for improving straw digestibility were studied in vitro, in situ, and in vivo. In Exp. 1, untreated straw (US); alkali-treated (5% NaOH, wt/wt) straw (AS); and autoclaved, alkali-treated straw (AAS) were sprayed with 0 or 1.5 mg/g DM of enzyme mix (xylanase, ß-glucanase, carboxymethylcellulase, and amylase) and incubated for 30 h in buffered ruminal fluid (3 x 2 factorial arrangement). Enzymes increased (P < 0.001) gas production and the incorporation of 15N into microbial N at 4 h, more so with AS or AAS than with US (P < 0.001 for gas; P < 0.05 for 15N). In Exp. 2, US and AS were sprayed with enzymes at 0, 0.15, or 1.5 mg/g DM (2 x 3 factorial) and incubated ruminally in nylon bags for up to 80 h to determine the in situ DM disappearance (ISDMD). Interactive effects (P < 0.05) of pretreatment and enzymes were observed on all ruminal degradation parameters. Alkali increased the rate (P < 0.01) and extent (P < 0.001) of ISDMD irrespective of enzymes. Enzymes applied to US did not affect the extent of ISDMD, but they increased (P < 0.01) the extent of ISDMD when applied to AS. Substrates from Exp. 1 and 2 were incubated in acetate buffer for 24 h to measure the hydrolytic loss of DM and release of reducing sugars and phenolic compounds. Alkali pretreatment and enzymes each increased (P < 0.001) DM loss and the release of reducing sugars and, in combination, exerted synergistic effects (P < 0.001). Enzymes did not affect the release of phenolic compounds from the straw. In Exp. 3, total-tract digestibility of untreated and enzyme-treated (100 mL/kg DM) ammoniated straw was assessed using 32 beef cows in eight pens. Wrapped straw bales were injected with NH3 (3% [wt/wt], DM basis) 4 mo before the study; enzymes were applied immediately before feeding. Applying enzyme to ammoniated straw increased (P < 0.05) digestibilities of DM, OM, and total N but did not affect the intake of DM or digestibility of ADF. Pretreatment of straw with alkali enhanced the efficacy of exogenous enzymes, presumably by breaking esterified bonds and releasing phenolic compounds and/or by swelling the crystalline cellulose and enhancing enzyme penetration. Including enzymes that mimic alkali hydrolysis (e.g., esterases) in commercial feed additives could substantially improve the value of these products for ruminants.
Key Words: Alkali Treatment • Digestion • Enzymes • Phenolic Compounds • Wheat Straw
Introduction
It is estimated that 2 x 109 t of straw are produced globally from cereal crops each year (FAO, 1996
). Although this agricultural by-product has a gross energy that is comparable to that of cereal grain, low digestibility limits its value as a feed source for ruminants. Considerable effort has been expended in an attempt to improve the feeding value of cereal straw (Fahey et al., 1993; Flachowsky et al., 1999), but in North America the majority of cereal straw is either reincorporated into the soil or used as bedding.
Recently, exogenous fibrolytic enzymes (EFE) have been evaluated for their potential to improve feed utilization in ruminants (Wang and McAllister, 2002a). Several of these studies have shown that these preparations can improve the rate of feed digestion (Nakashima and Ørskov, 1989; McAllister et al., 2000; Wang et al., 2002), but their ability to increase the extent of digestion may be limited by the lack of enzymes that degrade the core structure of lignin-cellulosic complexes. Several researchers have suggested that esterified bonds between cellulose, hemicellulose, and lignin restrict the digestion of recalcitrant cereal straws by ruminal microorganisms (Waghorn and McNabb, 2003). Alkali treatments, such as sodium hydroxide or ammonia, have been shown to be effective for cleaving esterified bonds within the plant cell wall architecture, enhancing enzymatic saccharification during fermentation (Gould, 1984) and improving the ruminal digestion of cereal straw (Sundstol, 1988); thus, it was hypothesized that alkali pretreatment may enhance the efficacy of EFE for improving the degradability of cellulosic feeds. This study was undertaken to determine whether such a synergistic relationship exists between alkali treatment and EFE, toward identifying factors or strategies to increase the use of straw in ruminant diets and to improve the value of EFE for ruminants.
Materials and Methods
In Vitro Study
An in vitro experiment with a 3 x 2 factorial arrangement of treatments was conducted to assess the effects of EFE on the ruminal fermentation of wheat straw that had been unmodified (control), treated with alkali, or treated with alkali and then autoclaved.
Wheat straw (3 kg total) was randomly selected from 40 large round bales at the Lethbridge Research Centre, ground through a 2.0-mm screen, and sieved with a 1.5-mm screen to isolate particles between 1.5 and 2.0 mm for use in the study. After thorough mixing, two-thirds of the ground straw was treated with 50% aqueous NaOH to achieve a final concentration of 5% NaOH (wt/wt, DM basis) on the straw. Half of this alkali-treated straw (AS) was held at room temperature in a sealed plastic bag for 24 h and then oven-dried overnight at 50°C. The other half of the AS was autoclaved (115°C, 20 min) immediately after mixing with alkali solution (AAS). The remaining third of the ground straw was treated with the same volume of water as used in the NaOH treatment, dried in the same manner, and used as the control (unmodified straw, US).
Each preparation of straw (US, AS, and AAS) was divided into two equal portions for treatment with an aqueous solution of EFE or an equal volume of deionized water (control). The enzyme preparation was a powdered 2:1 (wt:wt) combination of xylanase and ß-glucanase preparations from Trichoderma longibrachiatum (Biovance Technologies Inc., Omaha, NE) that exhibited (expressed as micrograms of reducing sugars [RS] released/[min•mg DM]) xylanase, ß-glucanase, carboxymethylcellulase, and amylase activities of 6.87, 5.00, 3.09, and 1.90, respectively (Wang et al., 2003). Straw was sprayed with deionized water or EFE solution at a rate of 10.0 mL/100 g of straw DM using separate single-nozzle bottle sprayers, to achieve final concentrations of 0 or 1.5 mg EFE/g straw DM. All the treatments were held in covered containers at room temperature for 24 h, and then stored at 4°C for 2 d before the in vitro incubation. Samples of the substrates were collected immediately before the incubation and transferred to storage at -40°C until analysis for chemical composition to enable estimation of DM loss and release of RS during the incubation.
The in vitro incubation was conducted in 35-mL serum vials as described by Wang et al. (2002) using buffered ruminal fluid as inoculum. Ruminal fluid was obtained from two steers fed a diet containing 50% wheat straw, 30% barley grain, and 20% alfalfa hay (DM basis), plus a mineral/vitamin supplement. Fluid was collected 1 h after the morning feeding from four locations in the rumen of each steer and strained through four layers of cheesecloth, and equal volumes of fluid from each steer were combined. Enriched ammonium sulfate (minimum 15N enrichment 10.01 atom percent excess, Sigma Chemical Company, St. Louis, MO) was included in the inoculum (1.5 g/L) to allow estimation of 15N incorporation into microbial N. Triplicate vials (300 mg substrate + 20 mL inoculum) of each treatment, plus controls (blanks) containing no substrate, were prepared for sampling at 4 and 30 h. Immediately before inoculation, 200 µL of 12% (wt/vol) NaOH was added to each blank as well as to those vials containing substrate US (no alkali treatment), in order that the amounts of sodium hydroxide in all vials would be similar.
Gas production in each vial was measured using a water displacement apparatus (Fedorak and Hrudey, 1983) eight times over the 30-h incubation period. After 4 and 30 h, triplicate vials were removed from the temperature-controlled chamber, and mercuric chloride was added to a final concentration of 0.02% (wt/vol). The entire contents were centrifuged (25,000 x g, 30 min, 4°C), and subsamples of supernatant were prepared for the determination of concentrations of VFA, RS, and total phenolic compounds (PC). Samples for RS determination were placed immediately in a boiling water bath for 15 min to inactivate enzymes; samples for VFA and PC were stored at -40°C. Incubation residues were washed three times by suspension in phosphate buffer (0.2 M, pH 7.4) followed by centrifugation (20,000 x g; 30 min; 4°C). The pellets were dried at 50°C, weighed for DM determination, and ground using a Pulverisette 7 planetary micro-mill (Laval Lab Inc., Laval, QC) for measurement of total N and 15N enrichment.
In Situ Study
The effect of alkali treatment and EFE on in situ DM disappearance (ISDMD) from wheat straw was assessed in nylon bag study with a 2 x 3 factorial arrangement of treatments (untreated vs. alkali-treated straw; EFE applied at 0, 0.15, and 1.5 mg/g straw DM).
Wheat straw was collected, ground, sieved, and treated with NaOH as described for the in vitro study, except that following the application of water or NaOH, the straw was held in plastic bags at room temperature for 5 d before oven drying (50°C). The EFE preparation and application method were also the same as in the in vitro experiment, except that two concentrations of EFE were studied. The resulting six treatments were kept in separate covered containers at room temperature for 3 h, sampled for determination of DM loss and release of RS, and then stored at 4°C for 2 d before use in the in situ experiment.
The nylon bag experiment was conducted using a 3-yr-old heifer with a permanent ruminal cannula. The heifer was fed a total mixed ration containing (DM basis) 68% barley silage, 12% chopped alfalfa hay, and 20% barley straw, plus mineral/vitamin supplement, for 14 d before initiation of the nylon bag experiment. Each of the substrates (5.0 g, as-treated basis) was weighed into monofilament nylon bags (12 x 15 cm; 50-µm pore size) and triplicate bags of each substrate were ruminally incubated for 0, 1.5, 6, 9, 16, 32, 56, and 80 h. Incubation, washing, and sample preparation procedures were those described by Wang et al. (1997).
Hydrolytic Incubations
Incubations of substrates in acetate buffer were conducted in conjunction with the in vitro and in situ studies to determine hydrolytic losses of DM, RS, and PC (in situ substrates only). Straw (250 mg) was weighed into 20-mL glass tubes, followed by 10.0 mL of 0.1 M acetate buffer (pH 4.5) with 0.01% (wt/vol) sodium azide added as an antimicrobial agent. The tubes were capped and incubated at 39°C for 24 h with shaking. Upon termination of the incubation, the mixtures were filtered through preweighed cellulose acetate membrane filters (0.45-µm pore size), and subsamples of filtrate were analyzed for RS (in vitro substrates) or RS and PC (in situ substrates) as described below. Residues retained on the filters were washed three times with deionized water (20 mL) and dried (along with the filters) for estimation of DM loss. Residues from the in vitro study substrates were dried at 105°C for 12 h, whereas those from the in situ study substrates were freeze-dried to facilitate analysis for PC as described below. All determinations were conducted in triplicate, on three consecutive days.
In Vivo Study
The effects of EFE on digestibility of alkali-treated wheat straw were investigated in a 33-d digestibility experiment conducted using 32 Continental x British crossbred beef cows (649.9 ± 11.9 kg) in late gestation.
Wheat straw was baled (2.15 m x 0.92 m x 0.02 m bales; average weight 225 kg) and stacked in early morning (85 to 88% DM), and covered with polyethylene sheeting within 24 h of baling. Anhydrous ammonia was injected into the stack to a final concentration of 3% (wt/wt), estimated by change of weight of the tank. The injection was conducted over three 8-h periods commencing 7 d after wrapping, by way of a closed-end, perforated, 2.5-cm (i.d.) steel pipe embedded in the stack. Application of ammonia was conducted over three consecutive days to avoid pressure build-up and possible escape of NH3. Anhydrous ammonia, rather than NaOH, was selected as the alkali treatment as it was easily injected and allowed for gaseous distribution of the treatment within the bales. The wrapped, injected bales were left undisturbed for 4 mo after ammoniation and then were opened and exposed to the air for 14 d before commencing the feeding trial.
The 32 cows were blocked by weight and randomly allocated to eight pens (5 m x 22 m), each equipped with a concrete floor, a three-sided shelter at one end, a stock waterer, and four individually covered feed bunks. The equivalent of 10 kg DM of ammoniated straw, 2.7 kg of alfalfa hay, and 100 g of mineral and vitamin supplement per cow (as-fed basis) was offered at 1500. The alfalfa hay was 89.2% DM, and its CP, ADF, NDF, Ca, and P contents (%, DM basis) were 16.8, 36.2, 48.7, 1.36, and 0.28%, respectively. The mineral and vitamin supplement comprised (as fed) 48.1% ground barley, 15.35% dicalcium phosphate, 8.00% Dynamate (Pitman-Moore Inc., Oakville, ON), 12.04% mineral mix, 9.33% vitamin premix, and 7.22% salt. The Dynamate contained 11% Mg, 18% K, and 22% S. The mineral mix contained (per kilogram) 6,000 mg Zn, 5,000 mg Mn, 2,000 mg Cu, 200 mg I, 50 mg Se, and 50 mg Co. The vitamin premix contained (per kilogram) 1,100 KIU vitamin A, 10.2 KIU vitamin D, and 616 IU vitamin E. In each pen, the straw was distributed evenly among the four feed bunks, treated as described below with water or EFE (four pens each), and then the alfalfa hay and supplement were top-dressed onto the straw. Feed residues were collected daily immediately before feeding and were weighed and dried to allow determination of DM intake on a per-pen basis. The alfalfa hay and supplement were completely consumed each day; orts consisted solely of straw.
The EFE preparation used in the study (Biozyme 411; Finnfeeds International Ltd., Marlborough, Wiltshire, U.K.) contained mainly xylanase (7,940 IU/g) and ß-glucanase (2,790 IU/g) activities. Immediately before feeding, 14 mL of EFE preparation was combined with 986 mL of water and the solution was sprayed onto the ammoniated straw (250 mL per feed bunk), yielding a final application rate of 386 mL/t of straw DM. Straw in the four pens designated as controls was similarly treated with 1 L of water.
Ammoniated straw, alfalfa hay, and feed residues were sampled daily from each feed bunk and composited for each pen. Fecal samples from the rectum were collected from each cow on d 29 to 33 of the experimental period and composited for each animal. All samples were stored at -40°C for subsequent chemical analysis. Digestibility was estimated using acid-insoluble ash (AIA) as an indigestible marker (Sunvold and Cochran, 1991).
All cattle used in the in vitro, in situ, and in vivo studies were cared for according to the guidelines set by the Canadian Council on Animal Care (CCAC, 1993).
Chemical Analyses
Concentrations of RS in substrates used in the in vitro study were estimated by weighing 100 mg of substrate into triplicate 20-mL screw-capped glass tubes and adding 10 mL of 0.05% (vol/vol) HCl. The tubes were capped, placed in a boiling water bath for 15 min to inactivate enzymes, and then placed on ice for 14 h. Following centrifugation (10,000 x g, 10 min, 4°C), the RS concentration in the supernatant was determined colorimetrically (Nelson, 1944).
Concentrations of PC were determined in the supernatant from cultures withdrawn from the in vitro incubation after 4 and 30 h, and in the filtrate and residues from the 24-h incubations of in situ substrates in the hydrolysis buffer. Liquid samples were centrifuged (10,000 x g; 10 min, 4°C) and the supernatants were combined with 4 vol of methanol (99.8%, Sigma Chemical Co.). After incubation for 30 min in the dark at 4°C, they were centrifuged again (10,000 x g; 10 min, 4°C) and the optical density of the supernatants at 280 nm was measured (Spectronic 1001 Plus, Milton Roy, New York, NY).
Phenolic compounds in the hydrolysis residues were measured using a modification of the method described by Lau and Van Soest (1981). Briefly, freeze-dried residues were ground for 5 min using a Pulverisette 7 planetary micro-mill, weighed into glass test tubes, and mixed with 0.1 M NaOH. The tubes were screw-capped and incubated for 20 h at room temperature, in the dark, with shaking. Aliquots of the mixture were combined with 1.2 vol of 0.1 M HCl and centrifuged (1,000 x g; 20 min, 4°C). The supernatants were diluted with 4 vol of methanol, and optical density was measured at 280 nm as described above. Ferulic acid (Sigma Chemical Co.) dissolved in methanol was used as standard; thus, PC contents are expressed as ferulic acid equivalents.
Feed, feed residues, and fecal samples from the in vivo study were dried at 55°C in a forced-air oven, whereas samples from other studies were freeze-dried. All samples were ground through a 1.0-mm screen before chemical analysis. Dry matter, OM, total N, NDF, VFA, and 15N enrichment in residual N were determined as described by Wang et al. (2002). All incubation liquids, reagent volumes, and rinsings generated subsequent to the preservation of subsamples with HgCl2 during the analysis of in vitro samples for PC, RS, and VFA were collected and stored in liquid waste containers for transport by a commercial waste management company to the Alberta Special Waste Treatment Facility at Swan Hills, AB. Concentrations of AIA and ADF in feed and fecal samples were determined using the methods described by Van Keulen and Young (1977) and by Komarek and Sirois (1993).
Calculation and Statistical Analyses
Gas production and the incorporation of 15N into microbial N during the in vitro ruminal study were calculated and expressed on the basis of substrate DM. In situ DM disappearance was estimated gravimetrically on the basis of substrate weights before and after ruminal incubation. Ruminal degradation parameters were calculated using the equations described by McDonald (1981):
 |
where P = DM degraded at time t (%), a = the soluble fraction (%), b = the potentially degradable fraction (%), c = the rate at which b is degraded (%/h), t = time (h) incubated in the rumen, and L = lag time (h). Effective degradability (ED) was calculated as
 |
with a, b, c, and L as described above and k = the ruminal outflow rate (%/h), which was set arbitrarily at 0.03. The constants a, b, c, and L for each animal were calculated using nonlinear regression procedures of SAS (SAS Institute Inc., Cary, NC).
Dry matter losses during the incubations in acetate buffer were expressed as a percentage of the substrate DM that was incubated. Relationships between the rate of EFE application and DM loss, RS release, and PC release during incubation of in situ substrates in acetate buffer were established by fitting the curves to the following nonlinear exponential equation:
 |
where Y = DM loss, concentration of RS, or concentration of PC; x = the rate of EFE application; and a0, a1, or a2 = calculated constant or coefficients. Relationships between the amount of PC remaining in the residue and the release of PC or RS as a function of DM loss were also established by linear curve fitting. All the curve fittings were performed using average values and SlideWrite Plus software (Advanced Graphics Software, Inc., Encinitas, CA).
Intake of DM during the in vivo study was calculated on a per-pen basis, as the difference between feed offered and feed refusals, and averaged across the entire experimental period. Acid-insoluble ash was used as indigestible marker for calculating the digestibilities of feed DM and of individual components OM, total N, and ADF according to the following:
 |
and
 |
where DMD = digestibility of DM (%); AIAfeed and AIAfeces = concentrations of AIA in the feed and feces (mg/g DM), respectively; CD = digestibility (%) of individual component C (where C = OM, total N, or ADF); and Cfeces and Cfeed = concentrations of component C in feces and feed (mg/g DM), respectively.
Data from the in vitro and in situ studies were analyzed as 3 x 2 and 2 x 3 factorial designs, respectively, and data from the in vivo study were analyzed as completely randomized design, using the Proc MIXED procedure of SAS (SAS Institute Inc., Cary, NC). Average values from triplicates in each of the three determinations were used for statistical analysis of data on DM loss and the release of RS during incubations of in vitro and in situ substrates in buffer. Mean values of four animals from each pen were used as the unit for statistical analysis of data from the in vivo study. Differences among the treatments were determined using contrast statements (SAS Institute Inc.). In all analyses, significance was declared at P < 0.05.
Results
In Vitro Study
The pretreatment of straw with alkali reduced (P < 0.001) OM, NDF, and RS contents but did not affect total N, as compared with untreated straw (Table 1). Enzyme application did not affect concentrations of OM, NDF, or total N but increased (P < 0.01) the RS content of the straw. Compared to alkali alone, alkali plus autoclaving reduced (P < 0.01) RS concentration but did not affect OM, total N, or NDF content of the straw. No interactive effects of pretreatment and EFE on nutrient concentrations were observed. However, pretreatment and EFE application exerted main (P < 0.01) and interactive (P < 0.01) effects on the loss of DM and release of RS from these substrates during the 24-h incubation in acetate buffer (Table 1). Pretreatment and EFE both increased (P < 0.01) DM loss and RS release. However, only RS release was increased (P < 0.001) by alkali plus autoclaving, as compared with alkali alone. The responses in DM loss and RS release were greater (P < 0.01) when the EFE were applied to pretreated than to untreated cereal straw.
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). With no EFE, gas production during the first 8 h was higher (P < 0.05) from AAS than from AS or US, which were similar. Applying EFE, however, increased (P < 0.01) gas production from AS relative to US at 4, 6, and 8 h, with both remaining lower (P < 0.01) than AAS. At 24 and 30 h, gas production from AS and AAS did not differ, and both were higher (P < 0.01) than US.
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). At 4 h, the incorporation of 15N was similar between AAS and AS, and both were higher (P < 0.01) than US, irrespective of EFE application (Table 3). Also, EFE had increased (P < 0.01) 15N incorporation for the alkali-treated substrates, but not for US. By 30 h, however, the EFE-mediated increases with AAS and AS were no longer evident, although 15N incorporation remained higher (P < 0.01) with AS and AAS than with US.
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With PC and total VFA concentrations, no interactive effects of pretreatment and EFE were observed. Concentrations of PC were higher (P < 0.01) with AS and AAS than with US throughout the incubation, and the difference was more pronounced at 4 h than at 30 h. However, treatment AAS increased PC and total VFA concentrations relative to AS only at 4 h (P < 0.001 and P < 0.01, respectively). Application of EFE increased (P < 0.01) VFA concentration only with AAS and only at 4 h. Total VFA concentration was generally higher (P < 0.01) with AAS than with US. Molar proportions of VFA in the incubation liquid were unaffected by alkali pretreatment or by EFE (data not shown).
In Situ Study
The loss of DM during the 24-h hydrolysis incubation was higher (P < 0.001) from alkali-treated straw than from the untreated straw (Figure 1). Application of EFE increased (P < 0.001) DM loss from both substrates. An interactive effect (P < 0.001) of alkali pretreatment and EFE application on DM loss was observed. The EFE-mediated increase in DM loss was greater (P < 0.001) for alkali-treated straw than for control straw. This pattern of alkali and EFE effects was mirrored in measurements of RS release (per gram of substrate DM). With PC, however, release was again greater (P < 0.001) with alkali pretreatment than without, but having applied EFE did not further enhance this effect.
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), and the rates of these increases were similar (P = 0.137) between substrates. In contrast, release of PC decreased as the EFE-related DM loss increased, and the rate of this decrease was greater (P < 0.001) for alkali-treated straw than for control. The proportion of PC in incubation residues increased as DM loss increased, and the rate of increase in the amount of PC retained per unit of DM loss was greater (P < 0.001) for alkali-treated straw than for control straw.
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), and an interactive effect (P < 0.01) of alkali treatment x EFE application on ISDMD was observed. Applying EFE increased (P < 0.01) ISDMD from alkali-treated straw but not from control straw. Percentages of ISDMD from alkali-treated straw were similar across EFE application levels (0.15 vs. 1.5 mg EFE/g DM).
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). Interactive effects of alkali pretreatment x EFE application were observed on fractions a (P < 0.01) and b (P < 0.05), on the rate (c) at which b was degraded (P < 0.001), lag time (P < 0.05), and on effective degradability (P < 0.05). Enzymes applied to untreated straw at 1.5 mg/g DM increased (P < 0.05) soluble fraction a but did not affect parameters b, c, lag, or ED, as compared with control (no EFE). In contrast, applied at 0.15 mg/g DM, EFE increased (P < 0.05) fraction a and reduced (P < 0.05) degradation rate c but did not affect parameters b, lag, or ED. When EFE were applied to alkali-treated straw, however, both application levels reduced (P < 0.001) lag time and increased (P < 0.05) ED, but a and b were unaffected.
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). Digestibilities of DM, OM, and total N in EFE-treated ammoniated straw were higher (P < 0.05) than those of untreated (no EFE) ammoniated straw. Enzyme treatment did not affect the intake of DM or OM, although cows consuming the EFE tended (P = 0.08) to have higher digestible DM intake than those that were not supplemented (data not shown).
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Exogenous fibrolytic enzymes have typically been observed to increase the initial rate but not the extent of DM digestion when used in ruminant diets (Varel et al., 1993; Feng et al., 1996; Wang and McAllister, 2002b). In the present study, however, applying EFE to straw following alkali treatment significantly increased both the rate and extent of DM digestion. The mechanism by which alkali treatment improved the efficacy of exogenous enzymes for enhancing feed digestion remains unknown, but findings from this study suggest that removal of the phenolic barriers that impede the microbial digestion of the feed may be involved.
Incorporation of 15N into feed particle-associated and non-feed particle-associated microbial N was increased by EFE at 4 h but not after 30 h of in vitro incubation. This is consistent with our earlier observations of exogenous enzymes enhancing microbial growth and colonization of feed particles by ruminal bacteria (Wang et al., 2001; Wang and McAllister, 2002b). The efficacy of sodium hydroxide and ammonia treatment for enhancing the activity of ruminal microorganisms is well documented (Kerley et al., 1985; Adebowale et al., 1989; Wang and McAllister, 2002b). The enzyme-mediated increase of 15N incorporation into microbial N was greater with alkali-treated as compared with control (untreated) straw, suggesting a synergistic effect of alkali pretreatment and EFE application on microbial activity in the rumen. This synergism probably arises from the combination of the alkali-mediated removal of structural barriers to rumen microbial colonization and EFE-mediated greater availability of soluble sugars for microbial growth (Wang and McAllister, 2002b).
Free phenolic acids and soluble phenolic-carbohydrate complexes have been shown to inhibit rumen microbial activity (Jung and Sahlu, 1986; Hartley and Akin, 1989; Martin and Blake, 1989), and there is evidence that these compounds are concentrated on the surface of feed particles during microbial digestion (Chesson et al., 1982). The present study demonstrated that the proportion of phenolic compound in the feed particles also increased during enzymatic hydrolysis. Alkali treatment (such as with NaOH or NH3) cleaves esterified bonds within the lignin-carbohydrate complex, reducing the physical enmeshment of cellulose and solubilizing the inhibitory phenolic compounds and, consequently, facilitating enzyme access (Chesson, 1981; Fahey et al., 1993) and microbial colonization of plant cell walls (Kerley et al., 1985). This study showed that alkali treatment significantly increased the amount of phenolic compounds, but not soluble carbohydrates, released from straw particles. In contrast, exogenous enzymes increased the release of soluble carbohydrates but not the release of phenolic compounds from straw particles. We have reported previously that supplementary soluble carbohydrates promoted the colonization of ruminal bacteria onto straw particles (Wang and McAllister, 2002b), as has been observed by other researchers (Barrios-Urdaneta et al., 2000). It was proposed that exogenous enzymes hydrolyzed the most digestible carbohydrates at the plant cell wall surface, and that accumulation of the lignin-carbohydrate complexes at the digestive surface impeded microbial colonization. Applying EFE onto straw following alkali pretreatment, therefore, synergistically integrated the positive effects of both treatments on enhancing microbial digestion of the straw.
This study showed that alkali pretreatment is more effective than EFE at enhancing microbial protein synthesis (in vitro data) and increasing digestion of straw (in situ data). This finding, combined with the observation that alkali pretreatment was more efficient than EFE for releasing PC from straw, suggests that the ability of exogenous enzyme preparations to cleave the esterified bonds within PC-mediated lignin-carbohydrate complexes may be limited, and, therefore, their effect on the extent of feed digestion may also be limited. This study demonstrated that, by applying these two treatments in sequence, their positive effects can be combined additively to increase straw digestion. The lignin-carbohydrate complex also contains ether bonds, but autoclaving the AS to cleave these bonds in addition to the alkali-mediated cleavage of the esterified bonds did not appear to further increase the incorporation of 15N into microbial N or DM digestion above what had been achieved by alkali treatment alone. Thus, screening new EFE preparations for esterase activity or adding such activity to existing preparations may be effective for substantially improving the value of these feed additives for ruminant diets. Likely, if such enzyme preparations were developed, they would be more effective with monocotyledonous than with dicotyledonous forages, in which the role of lignin-carbohydrate complexes in dictating cell wall digestion is less pronounced (Ben-Ghedalia et al., 1982).
The larger soluble and degradable fractions, shorter lag time, and increased ruminal effective degradability of alkali-treated straw compared with untreated straw were consistent with the general trend of the effect of alkali treatment (Fahey et al., 1993). Exogenous enzyme-mediated increases in RS concentration (at 0 h; Table 1
) and in soluble fraction (a) observed in untreated (but not alkali-treated) straw are indicative of preruminal enzymatic hydrolysis of straw DM, in accordance with our findings from earlier studies (Wang et al., 2001, 2002). The lack of EFE effects on soluble fraction and RS content in alkali-treated straw is likely due to the high pH of the alkali-treated straw, impeding maximal enzyme activity (Morgavi et al., 2001). However, a significant increase in the effective ruminal degradability of alkali-treated straw in association with EFE application was observed in this study, and a similar trend was observed in another study (Wang and McAllister, 2002b), which indicates that the synergistic effect of alkali treatment and EFE application on straw digestion does occur in the more moderate pH of the ruminal environment.
Ammoniation has been shown to increase the DM digestibility of cereal straws by approximately 15% (Fahey et al., 1993). Although the digestibility of unammoniated wheat straw was not measured in the present study, the apparent total-tract digestibilities of DM and OM of the ammoniated straw were similar to those measured in other studies (Horton, 1978; Ørskov et al., 1983; Jewell and Campling, 1986); thus, it is probable that ammoniation effected a typical increase in digestibility in this study. Enhanced digestibility of EFE-treated ammoniated straw as compared with non-EFE ammoniated straw suggests that the EFE-alkali synergism is also occurring in the total digestive tract. This is further supported by the fact that Willis et al. (1980) and Adebowale and Nakashima (1992) reported similar effects of NaOH plus exogenous enzymes on rice straw digestion, whereas researchers applying EFE onto untreated straw reported no significant effects on digestion (Nakashima et al., 1988; Rai and Mudgal, 1996; Liu and Ørskov, 2000). Further research is required to define the ideal conditions for the alkali pretreatment and EFE application to optimize this synergistic interaction.
Implications
Exogenous enzymes increased both the rate and the extent of digestion of alkali-treated wheat straw in vitro and in situ, and increased the extent of wheat straw digestion in vivo. It is hypothesized that the synergistic effect of these treatments on the microbial digestion of straw arises from their integrated actions to remove phenolic compounds and disrupt lignin-carbohydrate complexes by alkali hydrolysis and to increase the availability of soluble carbohydrates by enzymatic action. Although the combination of alkali and enzymatic treatment of straw may not be economically viable at the present time, including enzymes in commercial products that mimic alkali hydrolysis (e.g., esterases) might substantially improve the value of these products for ruminants.
Footnotes
1 This study was conducted with financial support from the Alberta Agricultural Research Institute, from the Matching Investment Initiative of Agriculture and Agri-Food Canada, and from Finnfeeds International, U.K. The authors gratefully acknowledge the assistance of Z. Xu, R. Coleman, and K. Jakober. This is Lethbridge Research Centre Contribution No. 38701045. 
2 Correspondence: P. O. Box 3000 (phone: 403-317-2240; fax: 403-382-3156; e-mail: mcallistery{at}agr.gc.ca).
Received for publication April 4, 2003. Accepted for publication September 11, 2003.
Literature Cited
Adebowale, E. A., and Y. Nakashima. 1992. Rumen degradation of some Leguminosae and Graminae roughages: Effect of chemical pre-treatment with or without cellulase preparation on dry matter and cell wall disappearance. Anim. Feed Sci. Technol. 38:219–235.
Adebowale, E. A., E. R. Ørskov, and P. M. Hotten. 1989. Rumen degradation of straw. 8. Effect of alkaline hydrogen peroxide on degradation of straw using either sodium hydroxide or gaseous ammonia as source of alkali. Anim. Prod. 48:553–559.
Barrios-Urdaneta, A., M. Fondevila, J. Balcells, C. Dapoza, and C. Castrillo. 2000. In vitro microbial digestion of straw cell wall polysaccharides in response to supplementation with different sources of carbohydrates. Austral. J. Agric. Res. 80:393–400.
Ben-Ghedalia, D., G. Shefet, J. Miron, and Y. Dror. 1982. Effect of ozone and sodium hydroxide treatments on some chemical characteristics of cotton straw. J. Sci. Food Agric. 33:1213–1218.
CCAC. 1993. Guide to the Care and Use of Experimental Animals. Vol. 1. 2nd ed. E. D. Olfert, B. M Cross, and A. A. McWilliam, ed. Canadian Council on Animal Care, Ottawa, ON.
Chesson, A. 1981. Effects of sodium hydroxide on cereal straws in relation to the enhanced degradation of structural polysaccharides by rumen microorganisms. J. Sci. Food Agric. 32:745–758.
Chesson, A., C. S. Stewart, and R. J. Wallace. 1982. Influence of plant phenolics acids on growth and cellulolytic activity of rumen bacteria. Appl. Environ. Microbiol. 44:597–603.
FAO. 1996. Production Year Book. Vol. 51. Food and Agriculture Organization of the United Nations, Rome, Italy.
Fahey, G. C., L. D. Bourquin Jr., E. C. Titgemeyer, and D. G. Atwell. 1993. Postharvest treatment of fibrous feedstuffs to improve their nutritive value. Pages 715–766 in Forage Cell Wall Structure and Digestibility. H. G. Jung, D. R. Buxton, R. D. Hatfield, and J. Ralph, ed. American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc., Madison, WI.
Fedorak, P. M., and S. E. Hrudey. 1983. A simple apparatus for measuring gas production by methanogenic cultures in serum bottles. Environ. Technol. Lett. 4:425–432.
Feng, P., C. W. Hunt, G. T. Pritchard, and W. E. Julien. 1996. Effect of enzyme preparations on in situ and in vitro degradation and in vivo digestive characteristics of mature cool-season grass forage in beef steers. J. Anim. Sci. 74:1349–1357.[Abstract]
Flachowsky, G., D. N. Kamra, and F. Zadrazil. 1999. Cereal straws as animal feed: Possibilities and limitations. J. Appl. Anim. Res. 16:105–118.
Gould, J. M. 1984. Alkaline peroxide delignification of agricultural residues to enhance enzymatic saccharification. Biotechnol. Bioeng. 26:46–52.
Hartley, R. D., and D. E. Akin. 1989. Effect of forage cell wall phenolic acids and derivatives on rumen microflora. J. Sci. Food Agric. 49:405–411.
Horton, G. M. J. 1978. The intake and digestibility of ammoniated cereal straws by cattle. Can. J. Anim. Sci. 58:471–478.
Jewell, S. N., and R. C. Campling. 1986. Aqueous ammonia treatment of wheat straw: Voluntary intake and digestibility in cattle. Anim. Feed Sci. Technol. 14:81–89.
Jung, H.-J. G., and T. Sahlu. 1986. Depression of cellulose digestion by esterified cinnamic acids. J. Sci. Food Agric. 37:659–665.
Kerley, M. S., G. C. Fahey, L. L. Berger, and F. Lee Baker. 1985. Alkaline hydrogen peroxide treatment unlocks energy in agricultural by-products. Science 230:820–822.
Komarek, A. R., and P. K. Sirois. 1993. Improved efficiency of ADF analysis using a filter bag procedure. J. Anim. Sci. 71(Suppl. 1):284.
Lau, M. M., and P. J. Van Soest. 1981. Titratable groups and soluble phenolic compounds as indicators of the digestibility of chemically treated roughages. Anim. Feed Sci. Technol. 6:123–131.
Liu, J. X., and E. R. Ørskov. 2000. Cellulase treatment of untreated and steam pre-treated rice straw: Effect on in vitro fermentation characteristics. Anim. Feed Sci. Technol. 88:189–200.
Martin, S. A., and G. G. Blake. 1989. Effect of phenolic compounds on a commercial hemicellulase and two cellulases. Nutr. Rep. Int. 40:685–693.
McAllister, T. A., K. Stanford, H. D. Bae, R. J. Treacher, J. Baah, J. A. Shelford, and K.-J. Cheng. 2000. Effect of a surfactant and exogenous enzymes on digestibility, growth performance and carcass traits of lambs. Can. J. Anim. Sci. 80:35–44.
McDonald, I. 1981. A revised model for the estimation of protein degradability in the rumen. J. Agric. Sci. 96:251–252.
Morgavi, D. P., V. L. Nsereko, L. M. Rode, K. A. Beauchemin, T. A. McAllister, A. D. Iwaasa, Y. Wang, and W. Z. Yang. 2001. Resistance of feed enzymes to proteolytic inactivation by rumen microorganisms and gastrointestinal proteases. J. Anim. Sci. 79:1621–1630.
Nakashima, Y., and E. R. Ørskov. 1989. Rumen degradation of straw. 7. Effects of chemical pretreatment and addition of propionic acid on degradation characteristics of botanical fractions of barley straw treated with cellulase. Anim. Prod. 48:543–551.
Nakashima, Y., E. R. Ørskov, P. M. Hotten, K. Ambo, and Y. Takase. 1988. Rumen degradation of straw. 6. Effect of polysaccharidase enzymes on degradation characteristics of ensiled rice straw. Anim. Prod. 47:421–427.
Nelson, N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153:375–380.
Ørskov, E. R., G. W. Reid, S. M. Holland, C. A. G. Tait, and N. H. Lee. 1983. The feeding value for ruminants of straw and whole-crop barley and oats treated with anhydrous or aqueous ammonia or urea. Anim. Feed Sci. Technol. 8:247–257.
Rai, S. N., and V. D. Mudgal. 1996. Effect of alkali and (or) steam treatment of wheat straw or cellulase augmented concentrate mixture on rumen fermentation in goats. Small Rumin. Res. 19:219–225.
Sundstol, F. 1988. Straw and other fibrous by-products. Livest. Prod. Sci. 19:137–158.
Sunvold, G. D., and R. C. Cochran. 1991. Technical note: Evaluation of acid detergent lignin, alkaline peroxide lignin, acid insoluble ash, and indigestible detergent fiber as internal markers for prediction of alfalfa, bromegrass, and prairie hay digestibility by beef steers. J. Anim. Sci. 69:4951–4955.[Abstract]
Van Keulen, J., and B. A. Young. 1977. Evaluation of acid-insoluble ash as a natural marker in ruminant digestibility studies. J. Anim. Sci. 44:282.
Varel, V. H., K. K. Kreikemeier, H. G. Jung, and R. D. Hatfield. 1993. In vitro stimulation of forage fiber degradation by ruminal microorganisms with Aspergillus oryzae fermentation extract. Appl. Environ. Microbiol. 59:3171–3176.
Waghorn, G. C., and W. C. McNabb. 2003. Consequences of plant phenolic compounds for productivity and health of ruminants. Proc. Nutr. Soc. 62:383–392.[Medline]
Wang, Y., and T. A. McAllister. 2002a. Rumen microbes, enzymes and feed digestion: A review. Asian-Australas. J. Anim. Sci. 15:1659–1676.
Wang, Y., and T. A. McAllister. 2002b. Investigation of exogenous fibrolytic enzyme activity on barley straw using in vitro incubation. J. Anim. Sci. 80(Suppl. 1):316.
Wang, Y., T. A. McAllister, D. R. ZoBell, M. D. Pickard, L. M. Rode, Z. Mir, and K.-J. Cheng. 1997. The effect of micronization of full-fat canola seed on digestion in the rumen and total tract of dairy cows. Can. J. Anim. Sci. 77:431–440.
Wang, Y., T. A. McAllister, L. M. Rode, K. A. Beauchemin, D. P. Morgavi, V. L. Nsereko, A. D. Iwaasa, and W. Yang. 2001. Effects of an exogenous enzyme preparation on microbial protein synthesis, enzyme activity and attachment to feed in the Rumen Simulation Technique (Rusitec) 85:325–332.
Wang, Y., T. A. McAllister, L. M. Rode, K. A. Beauchemin, D. P. Morgavi, V. L. Nsereko, A. D. Iwaasa, and Y. Wang. 2002. Effects of exogenous fibrolytic enzymes on epiphytic microbial populations and in vitro silage digestion. J. Sci. Food Agric. 82:760–768.
Wang, Y., T. A. McAllister, J. Baah, R. Wilde, K. A. Beauchemin, L. M. Rode, J. A. Shelford, G. M. Kamande, and K.-J. Cheng. 2003. Effects of Tween 80 on in vitro fermentation of silages and interactive effects of Tween 80, monensin and exogenous fibrolytic enzymes on growth performance by feedlot cattle. Asian-Australas. J. Anim. Sci. 16:968–978.
Willis, C. M., O. T. Stallcup, and D. L. Kreider. 1980. Influence of sodium hydroxide and enzyme additions on nutritive values of rice straw. J. Anim. Sci. 50:303–308
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