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* Faculty of Agricultural Science, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
Sustainable Production Systems, Research Center, Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada T1J 4B1
Corresponding Author:
K. A. Beauchemin; e-mail:
beauchemin{at}agr.gc.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Abbreviation key: CMC = carboxymethyl cellulose, CONC = enzyme product added to concentrate (45% of TMR DM basis), CTRL = control treatment, DDMI = digestible dry matter intake, PD = urinary purine derivative, PREM = enzyme product added to premix (0.2% of TMR DM basis), SUPP = enzyme product added to supplement (4% of TMR DM basis)
Key Words: dairy cows • enzymes • digestibility • application method
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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An increase in milk production was reported in some studies when dairy cow diets were supplemented with fibrolytic enzymes (Rode et al., 1999; Yang et al., 2000), but not in others (Beauchemin et al., 2000). Milk production is not the only inconsistent variable. DMI was reported to be both increased (Beauchemin et al., 2000) and unchanged (Beauchemin et al., 1999, Kung et al., 2000) when enzymes were added to the diet. Similarly, effects of supplemental enzymes on digestibility have been inconsistent. Use of enzyme products comprised mainly of xylanases and cellulases have been shown to increase digestibility (Rode et al., 1999; Yang et al., 2000), or have no effect on digestibility (Lewis et al., 1999). It is essential to determine the conditions necessary for supplemental fibrolytic enzymes to have beneficial effects on animal performance.
The objectives of this study were to compare the effects of applying a fibrolytic enzyme product on different proportions of the TMR on nutrient intake and digestibility, ruminal fermentation, microbial N synthesis, digesta passage kinetics, and milk production and composition.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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The design of the experiment was a double 4 x 4 Latin Square with each period lasting 28 d. Four of the cows were fitted with ruminal cannulas. Cows received a diet consisting of 45% concentrate and 55% forage (DM basis) (Table 1 and 2
). The concentrate contained steam-rolled barley and a pelleted supplement. The diet was formulated using the Cornell-Penn-Miner System (CPMDairy, Version 1.0) and balanced to provide sufficient metabolizable protein, metabolizable energy, vitamins, and minerals to produce 35 kg/d of milk with 3.5% fat and 3.2% CP. Presence or absence of a fibrolytic enzyme product and the proportion of TMR to which the enzyme product was added made up the four treatments. The treatments were: 1) no enzyme (CTRL), 2) enzyme added to the concentrate portion of the TMR consisting of 45% of the dietary DM (CONC), 3) enzyme added to the pelleted supplement portion of the TMR which made up 4% of the dietary DM (SUPP) and 4) enzyme applied to a premix, offered at 50 g/head/d, which made up 0.2% of the dietary DM (PREM).
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The appropriate amount of enzyme powder was dissolved in water, and then added at the time of milling. For the CONC treatment, the enzyme solution (93 g/20 L water) was added slowly into a 1-t mixer containing steam-rolled barley and pelleted supplement. For the SUPP treatment, the diluted enzyme (93 g/15 L) was added to 250 kg of ingredients in the mixer prior to pelleting the supplement. Due to the small volumes that were required for the PREM treatment, diluted enzyme (4 g/16 ml) was added to 200 g of wheat bran and mixed using a food processor. All three enzyme-feed mixtures were prepared at the beginning of each period. Treated feed was discarded at the end of each period to ensure that the time between applying exogenous enzymes to feed and feeding was the same for all treatments.
The enzyme product activities (Table 3) were assayed using carboxymethyl cellulose (CMC, medium viscosity, Sigma, St. Louis, MO); Avicel PH105 20 µm (FMC Corporation, Philadelphia, PA); oat spelt xylan (Sigma, St. Louis, MO); and wheat arabinoxylan, xyloglucan, and barley ß-glucan (Megazyme International Ireland Ltd., Wicklow, Ireland) as substrates. Assays were conducted by adding 50 µl of enzyme solution to a tube containing 100 µl of 0.1 M sodium citrate and phosphate buffer (pH 5.0 and 6.0) and 50 µl of 2% substrate. The contents were then incubated at 39°C for 10 min; the reaction was stopped with the addition of Somogyi reagent and boiling. Blanks were also used for corrections. Reducing sugars liberated from the hydrolysis of the various substrates were detected using the Nelson-Somogyi method (Somogyi, 1952). Acetyl-esterase (EC 3.1.1.6) was assayed with p-nitrophenyl substrates obtained from Sigma (St. Louis, MO). The assays were carried out in microtiter plates and consisted of 20 µl of diluted enzyme sample and 80 µl of 1 mM substrate in 0.1 M citrate and PO4 buffer (pH 5.0 and 6.0). The mixture was incubated at 39°C for up to 30 min; the reaction was then stopped with 100 µl of 0.5 M glycine and NaOH buffer, pH 10.6. Release of p-nitrophenol was measured at 420 nm using a MRX-HD plate reader (Dynatech Laboratories, Inc., Chantilly, VA).
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The first 11 days of each period were for adaptation, d 12 to 17 were used to determine rate of passage, d 12 to 14 for digestibility measurements, d 14 to 16 for urine collection, d 21 to 28 for milk yield and composition determination and d 26 for rumen fermentation measurements.
Feed offered and refused was measured and recorded daily to determine DMI. Barley silage and alfalfa silage was sampled weekly and DM was determined to adjust diet composition when required. The TMR and ort samples were collected daily for 1 wk coinciding with milk sampling. The TMR and ort samples were dried at 55°C and then ground to pass a 1-mm screen (standard model 4; Arthur H. Thomas Co., Philadelphia, PA) and composited for each cow by period and subsequently analyzed for NDF, ADF, and OM. Milk production was recorded daily and sampled morning and evening for 1 wk. Milk samples were preserved with potassium dichromate and stored at 4°C until sent to Central Alberta Milk Testing Laboratory (Edmonton, AB, Canada). Milk was analyzed for milk fat, CP, and lactose (AOAC, 1990) using an infrared analyzer (Milk-O-Scan 605; Foss Electric, Hillerød, Denmark).
Cows were weighed at the beginning and end of each period at approximately 0900 h; these weights were then used to calculate mean BW for each period.
Ruminal Fermentation and Rate of Passage
Ruminal fluid was collected from cannulated cows at 0730 and 1300 h. Samples were taken from four locations within the rumen, composited and then squeezed through four layers of cheesecloth with a mesh size of approximately 250 µm. Five milliliters of filtered rumen fluid was added to 1 ml of 25% HPO3 for VFA determination and 5 ml of filtrate was added to 1 ml of 1% sulfuric acid for NH3 determination. Samples were stored at –20°C until analysis.
Cr-mordanted NDF was used to measure the rate of passage of particulate matter and Co-EDTA was used as a liquid phase marker. Barley silage was boiled in detergent and rinsed until the NDF content of the residual fiber exceeded 85%. The resulting fiber was dried at 55°C. Chromium was mordanted to the barley silage fiber and Co-EDTA was prepared according to Udén et al. (1980).
Each cannulated cow received 225 g of Cr-mordanted barley silage and a 300 ml solution containing 15 g of Co-EDTA via the rumen cannula on d 11. Rectal samples of feces were collected at 0, 3, 6, 9, 12, 16, 20, 24, 28, 32, 36, 40, 48, 60, 72, 96 and 120 h after dosing. Samples were oven dried at 55°C and ground to pass a 2-mm screen (standard model 4) and stored until analysis.
Particulate and liquid kinetic parameters were estimated for each cannulated cow for each period from the concentration of Cr and Co, respectively in the feces. A double compartmental model (Grovum and Williams, 1973) was fitted using the non linear regression procedure of SAS (SAS, 1999).
Apparent Digestion
Apparent total tract digestion of nutrients was determined for all cows using YbCl3 (Rhône-Poulenc, Inc., Shelton, CT). Ytterbium solution was incorporated directly into the concentrate at the time of milling. Cows received approximately 1 g of Yb/cow daily throughout the entire study. Fecal samples were taken beginning on d 12 at 0900 h then 3, 6, 9, 12, 16, 20, 24, 28, 32, 36, 40, 48 and 60 h later. Samples were individually dried and ground to pass a 2-mm screen (standard model 4), composited across sampling times for each cow and stored for chemical analysis.
The concentration of Yb was analyzed in feed, orts and feces and then digestibility was calculated using the indicator method (Schneider and Flatt, 1975).
Microbial Nitrogen Synthesis
One kilogram of fresh rumen contents obtained from each cannulated cow during rumen evacuation the last day of each period was blended (Waring Products Division, New Hartford, CT) with 1 liter of 0.9% NaCl for 2 min and then squeezed through four layers of cheesecloth. The filtrate was centrifuged (800 x g for 15 min at 4°C) to remove feed particles and then the supernatant was further centrifuged (20,000 x g for 45 min at 4°C) to obtain a bacterial pellet. Bacterial pellets were freeze-dried and ground using a ball mill (Wig-L-Bug; Crescent Dental Mfg. Co., Lyons, IL). The samples were then stored for analysis of purines-N and total-N.
Total output of urine was collected from cannulated animals on d 14 to 16 using a balloon catheter (Rüsch Canada, North York, ON, Canada). Urine was collected in vessels containing sufficient amounts of 4 N H2SO4 to maintain pH below 3. Urine volume was measured daily and samples were kept at –20°C until analysis.
Total purine derivatives (PD) excreted (mmol/d) were estimated as the sum of uric acid and allantoin. Excretion of the endogenous PD was assumed to be constant at 0.385 mmol/kg BW0.75 for individual cows (Chen and Gomes, 1992). Purine absorption of microbial origin (mmol/d) was calculated as:
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Synthesis of microbial N within the rumen was calculated as:
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The purine-N and total-N were determined from bacterial pellets obtained from individual cows and the average purine N: total N measured in this study was 0.136.
Chemical Analysis
Analytical DM was determined by drying samples at 135°C for 3 h. Oven DM was determined by drying samples at 55°C for 48 h. Organic matter was determined by ashing (AOAC, 1990). The NDF and ADF contents were determined using an ANKOM200/220 Fiber Analyzer (ANKOM Technology, Fairport, NY) according to the methodology supplied by the company, which is based on the methods described by Van Soest et al. (1991). Sodium sulphite and heat-stable amylase were used in the analysis of NDF. The N content was determined by flash combustion (Carlo Erba Instruments, Milan, Italy). Contents of Co, Cr and Yb were determined using atomic absorption according to the AOAC procedure (AOAC, 1990). Ruminal VFA were separated and quantified using gas chromatography (5890; Hewlett Packard, Mississauga, ON, Canada) using a 30-m (0.32-mm i.d.) column (Nukol column; Supelco, Oakville, ON, Canada). Rumen ammonia was determined as described by Rhine et al. (1998).
Allantoin in urine was determined by autoanalyzer using the procedure of Pentz (1969) with modifications by Lindberg and Jansson (1989). Uric acid was determined using a commercial kit (Sigma No. 292; Sigma Chem. Co., St. Louis, MO). Purine content in the microbial pellet was determined using the procedure of Zinn and Owens (1986).
Statistical Analysis
Means were calculated for all variables by cow within period. Data were analyzed using the MIXED procedure of SAS (SAS, 1999). Period and cow were considered random effects; diet and cannulation effects were considered fixed. Estimation method was restricted maximum likelihood and the degrees of freedom method was Kenward-Roger (SAS, 1999). Differences were tested using the PDIFF option in SAS (SAS, 1999) using a protected (P < 0.10) LSD test. Differences were declared significant at a P < 0.05; and trends were discussed at a P < 0.15, unless stated otherwise.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). However, cows receiving the CONC treatment produced more milk than cows receiving the PREM treatment. Although not statistically significant, cows receiving the CONC treatment had numerically higher FCM; 3% more than the CTRL and SUPP treatments, and 5% more than the PREM treatments. Use of an enzyme supplement tended to increase both fat and protein percentage and decrease lactose percentage in milk, however only the PREM treatment was significantly different from CTRL.
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). There was a significant interaction between the main effects of treatment and cannulation for DMI. Cannulated cows receiving the CONC treatment decreased DMI in comparison to cannulated cows receiving CTRL, while their intact counterparts increased DMI while receiving the CONC treatment compared to CTRL (Figure 1). Cannulated cows receiving the SUPP and PREM treatments increased DMI compared to the CTRL. Dry matter intake for intact cows increased for SUPP and decreased for PREM in comparison to intact cows receiving the CTRL treatment.
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). Total tract digestion of NDF and ADF digestion was also increased in CONC fed cows compared to CTRL. Cows receiving SUPP and PREM treatments showed a nonsignificant, intermediate increase in nutrient digestibility compared with CTRL. Treatment differences in rumen pool size of the various constituents were not significant (Table 6).
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). The SUPP and PREM treatments had differing molar proportions of propionate postfeeding, but neither treatment differed from the control. The differences in propionate concentration led to differences in the acetate to propionate ratio between these two treatments. Ammonia nitrogen concentration in the rumen both pre- and postfeeding did not differ between treatments and was highly variable as shown by the large standard error.
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).
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). Cows fed PREM produced significantly more microbial nitrogen than did cows receiving the SUPP treatment, with intermediate levels of microbial nitrogen from cows fed CONC. However, microbial nitrogen from cows fed the enzyme treatments was not different (P > 0.05) from cows fed the control treatment. Although not statistically significant, the CONC and PREM treatments increased microbial nitrogen production by 6% and 9%, respectively, compared to the CTRL (P = 0.09).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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The application of the enzyme product prior to pelleting, as was the case for the SUPP treatment, appears to have hindered the efficacy of the product based on the lack of improvement in total tract digestibility. The pelleting process involved temperatures as high as 94°C, which may reduce the activity of this enzyme, thereby delivering insufficient activity levels when fed to the animal. Unfortunately we were not able to quantify the possible decline in enzyme activity due to pelleting because reliable methods for detecting exogenous enzymes added to feeds are lacking (McCleary, 2001). Methods that rely on extracting the enzymes from feeds are subject to the errors associated with inadequate enzyme recovery. Furthermore, the background reducing sugar content of supernatant due to the soluble feed substrate greatly increases the potential for errors (Wallace and Hartnell, 2001). Applying the enzyme product to the supplement after the pelleting process may be a viable solution for future studies. Alternatively, decreased efficacy of enzymes for the SUPP treatment may indicate that enzymes are more effective when applied to a larger portion of the diet, as was the case when enzymes were added to concentrate.
Providing the enzyme product to cows in a concentrated premix was also not effective based on the lack of improvement in total tract digestibility. The portion of the ration treated with enzyme for CONC was larger, in comparison to SUPP and PREM, which may have maximized the dispersion of the enzyme within the rumen and increased the likelihood of the exogenous enzyme being beneficial. Beauchemin et al. (1999) postulated that applying enzymes to feed creates a "slow release" mechanism releasing the enzyme into the rumen fluid as feed is digested. The decreased size of the premix may have allowed rapid passage out of the rumen, lessening the enzyme effect for cows fed PREM. This hypothesis is further supported by an in vitro study using the same treatments showing 18% and 20% increases in DM digestibility for CONC and PREM, respectively (unpublished). Thus, having the enzyme delivered in a highly concentrated form did not hinder the product in vitro, but residence time is not a factor in batch culture. In contrast, residence time of enzyme in the rumen may have been reduced in the present study for cows receiving PREM, thereby negating potential benefits.
It is possible that applying enzyme to concentrate increased digestion by enhancing the digestibility of the barley grain. It is well documented that the close association of enzyme with feed enables a form of preingestive attack of the enzymes upon the plant fiber (Wang et al., 2002). However, this explanation does not account for the increase observed in vitro for the premix treatment (unpublished). Thus, it is unlikely that the digestion response was dependant upon the substrate to which the enzyme was applied.
Previously it was thought that exogenous enzyme sources were susceptible to rumen microorganisms and gastrointestinal degradation. This notion has recently been refuted and it is now known that some exogenous enzyme sources have the ability to withstand both ruminal and intestinal degradation (Morgavi et al., 2001), although resistance to proteolysis is not the same for all enzyme products (Hristov et al., 1998a; Hristov et al., 1998b).
There are few studies in which cannulated and intact animals are studied simultaneously and the interaction between diet and cannulated effects for DMI were unanticipated (Figure 1). Cannulated cows decreased DMI when receiving CONC, however due to the increase in total tract digestibility, intake of DDM was increased for cows fed CONC compared to cows fed CTRL. It is hypothesized that due to production differences between cannulated and intact cows additional nutrients available due to enzyme supplementation were in excess of requirements and cannulated cows receiving the CONC treatment subsequently lowered DMI. Intake of DDM for cows fed PREM was similar to cows fed CTRL, indicating comparable total tract digestion.
Milk production and composition were reported, but were not the main focus of this study due to the limited number of cows used. Other researchers feeding various enzyme products have shown both an increase (Rode et al., 1999; Yang et al., 2000) and no change (Beauchemin et al., 2000) in milk production. In the present study, the numerical increase in 4% FCM for cows fed CONC was attributed to the increase in diet digestibility because DMI was not affected by diet. Milk production followed the same trend as intake of DDM. This experiment confirmed previous studies that showed enzyme supplementation did not increase DMI (Beauchemin et al., 1999; Hristov et al., 1998a; Kung et al., 2000). However, the effects of enzyme supplementation on DMI may depend on the specific formulation evaluated, as other studies using different enzyme products fed to beef steers (Feng et al., 1996) or dairy cows (Beauchemin et al., 2000) have shown an increase in DMI.
The trend of increased fat and/or protein percentage due to fibrolytic enzyme supplementation has been previously reported (Luchini et al., 1997; Nussio et al., 1997; Schingoethe et al., 1999). In contrast, Kung et al. (2000) showed similar fat and protein percentages with enzyme supplementation, while Beauchemin and coworkers reported an increase in protein percentage with no difference in daily production of fat or protein (Beauchemin et al., 2000). To date, most studies that reported an increase in milk production due to enzyme supplementation observed either a decrease or no effect on fat and protein percentage (Rode et al., 1999; Yang et al., 1999; Kung et al., 2000; Yang et al., 2000). In our study, it is hypothesized that the increased intake of digestible energy due to enzyme supplementation (CONC) that was not used for milk production was allocated to milk fat, milk protein, and body reserves. This is also supported by the numerical increase for change in BW for cows fed CONC compared to cows fed CTRL.
Despite the increase in total tract feed digestion, the response in milk production was not observed with enzyme supplementation. The positive energy status of the cows was attributed to the lack of response. However, calculated energy status revealed that cow receiving the CONC treatment had a larger surplus of energy after maintenance energy requirements were met. If this excess energy had been partitioned into milk and milk composition remained unchanged, then daily milk output would have increased 9% in cows receiving the CONC treatment in comparison to cows receiving CTRL. This implies that cows in early lactation and negative energy balance may benefit from receiving supplemental enzymes.
Though no difference was seen for digesta or passage kinetics in this study, others have reported trends of increased digesta passage for dairy cows (Beauchemin et al., 1999) and significantly increased digesta passage for beef steers (Feng et al., 1996) due to enzyme supplementation. However, fibrolytic enzyme supplements have generally been shown not to alter passage of either ruminal fluid or particulate material (Lewis et al., 1996; Yang et al., 1999; Hristov et al., 2000).
The decrease in propionate, subsequently leading to an increase in the acetate to propionate ratio was unexpected for the PREM treatment. Previous studies providing enzyme supplementation have reported no effect (Beauchemin et al., 1999; Kung et al., 2000) to slight differences in molar ratios (Hristov et al., 2000), even when the total VFA concentration was increased (Lewis et al., 1996). Differences in ammonia N concentrations pre- and postfeeding were expected and are explained by decreased synchrony between protein and energy.
Two main factors can affect rumen microbial yield: rate of outflow from the rumen and amount of OM fermented within the rumen. Because rumen flow kinetics were not affected by enzyme supplementation, this is not likely to account for increased microbial yield due to enzyme supplementation. The trend of increased microbial nitrogen synthesis is attributed to an increase in ruminal digestion and overall rumen efficiency.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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2 Dr. J. A. Shelford passed away on April 6, 2002.
Received for publication April 19, 2002. Accepted for publication July 19, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and intact (
) cows (SE=0.63) and (Bottom) digestible dry matter intake (DDMI) for cannulated (