Unlocking Fiber’s Real Potential...Do Enzymes Really work?

Enzymes are complex organic compounds comprised primarily of proteins. Their activity is due to a highly ordered three-dimensional structure that is specific for the substrate or molecules upon which the enzyme acts.

The structural affinity of a digestive enzyme for its substrate determines its velocity or rate of catabolism. Enzyme structural affinity can be modified by numerous environmental factors, including: moisture, pH, temperature, and proteolysis.

Some enzymes require the presence of coenzymes or cofactors. Some enzymes are constitutive in nature, that is, they are intimately associated with the organisms that produce them. Fibrolytic enzymes are an example of constitutive enzymes. Thus, cell-free ruminal fluid has very little fibrolytic activity.

Digestion is a metabolic process that occurs largely through the catabolic action of enzymes. Limitations on rate of digestion are a function of: 1) enzyme reactivity or velocity (enzyme:substrate affinity); 2) enzyme concentration; and 3) exposure rate of substrate to the enzymatic process.

In all, it should be clear that there are many ways to promote enzymatic activity and hence, the digestive processes of cattle. The objective of this presentation is to discuss the use of enzymes to enhance fiber digestion in ruminants.


Fiber Digestion

Plant cell walls are comprised of a complex array of carbohydrate fractions including hemicellulose, cellulose, and lignin that impart rigidity and structural stability needed for growth. These fractions are referred to collectively as neutral detergent fiber or NDF. Although cellulose is the predominant component of plant fiber, it is important to recognize that the cellulose microfibrils are tightly bound by covalent bonding in a matrix of other fiber components, particularly hemicelluloses and lignin (Jeffries, 1990). Analogous to reinforced concrete, digestion of cellulose is limited by this hemicellulose-lignin encasement.

Ruminants do not produce fibrolytic enzymes themselves, but depend entirely on their symbiotic relationship with microorganisms for the digestion of plant cell walls. Hemicellulose is composed of dense pentose polymers known as xylans. Fibrolytic bacteria hydrolyze the complex bundles of cellulose and xylans via extensive coordination or synergy between cellulase and xylanase enzymes that are held in close association as part of a marvelous fibrolytic complex (Tomme et al., 1995). During the course of digestion, hemicellulose and cellulose fractions are broken down into simpler sugars; lignin is indigestible.

Depending on hydration, cellulose crystalinity, and degree of cross linking, the digestion rates of plant fiber is quite variable. In stark contrast with starch digestion, ruminal digestion of pure cellulose, even under optimal conditions, is relatively slow, ranging between 3 to 12%/h (Mertens, 1992).

As stated previously, the fibrolytic enzymes are constitutive by nature. The fibrolytic enzyme complexes are directly connected to the microorganisms that produce them, and hence, ruminal factors that influence the growth and dispersion of ruminal fibrolytic organisms will have a direct effect on fiber digestion. Critical factors that are known to suppress growth of fibrolytic microorganisms include supplemental fat, ionophores, oral antibiotics, and ruminal pH.


Supplemental fat

The negative associative effects of supplemental fat on ruminal fiber digestion are well documented (Brethour et al., 1957; Davidson and Woods., 1960; Devendra and Lewis., 1974; Boggs et al., 1987; Zinn, 1988,1989, 1994; Zinn and Plascencia, 1993, 1996; Zinn et al., 2000).

Depression of ruminal fibrolytic capacity with fat supplementation typically ranges between 15 and 40%. The basis for the depression is not due to physical coating of feed particles (Zinn and Plascencia, 1996), but rather to the toxic effects of supplemental fat on ruminal fibrolytic organisms, particularly protozoa. In vitro studies (Henderson, 1973; Maczulak et al.,1981) demonstrate that the unsaturated fatty acids, particularly C18:1, play the more active role in inhibiting ruminal cellulolytics.


Supplemental ionophores

One of the more consistent effects of ionophore supplementation on digestive function has been a reduction in ruminal fiber digestion (Simpson, 1978; Poos et al., 1979; Zinn, 1987; Zinn and Borquez, 1993).


Oral antibiotics

Tetracycline antibiotics have been found to be strong inhibitors of cellulose digestion both in vitro (Simpson et al., 1976; Baldwin et al., 1982) and in vivo (Tollman and MacVicar, 1956; Evans et al., 1957; Zinn, 1992). As little as 22 mg of chlortetracycline/kg of diet has decreased total tract fiber digestion in cattle by as much as 5 to 15%. Zinn (1992) observed that supplementing a 29% forage receiving diet with 130 mg/kg tetracycline reduced ruminal fiber digestion in Holstein steers by 30%.


Ruminal pH

Ruminal pH affects fiber digestion through its influence on the specific growth rates of cellulolytic bacteria. Growth of cellulolytic bacteria is optimal at ruminal pH of greater then 6.5.

Between pH of 6.5 to 6.0, the specific growth rate decreases 14%/h for every .1 unit decrease in ruminal pH. Cellulolytic bacteria do not grow at ruminal pH below 6.0. This toxicity is due apparently to the inability of cellulolytic bacteria to regulate intracellular anion concentrations at lower ruminal pH (Russell and Wilson, 1996). Increasing the level of nonstructural carbohydrates (grain, molasses, bakery waste, etc.) in the diet, increasing level of feed intake, and/or increasing the level of grain processing are likely to depress ruminal pH.


Fiber digestion vs digestibility

The term “digestibility” is qualitative, referring to susceptibility to degradation. Whereas, the term “digestion” refers to the extent of degradation. The extent of ruminal fiber digestion is a function of rate of digestion (Kd) and rate of passage (K_p) [digestion = K_d/(K_d + K_p]. A primary factor that influences rate of ruminal fiber digestion is the accessibility of substrate to the fibrolytic process, in particular, the physical chemical interactions of cellulose, hemicellulose and lignin. This is a unique characteristic of individual forages or fiber sources, and is influenced by stage of maturity, age post harvesting, method of preservation, and processing.

Aside from the unique physical-chemical aspects of the fiber sources, rate of ruminal fiber degradation will be influence by the ruminal fibrolytic capacity. This capacity is a function of ruminal microbial distribution and adaptation. As stated previously, this capacity will be greatly influenced by ruminal environmental factors, including pH and presence of toxins or inhibitors.

The retention time of fiber in the rumen, or the length of time that fiber is exposed to the fibrolytic process is influenced by initial fiber particle size, rate of particle size reduction (chewing, rumination), particle density, and rate of digestion. The basis for particle retention is not altogether certain. Larger particles may become trapped in or on the floating ruminal mat, thus restricting access to the reticulo-omasal orifice. Larger particles may also be screened by omasal lamina and returned to the rumen, increasing retention time of larger particles (Church, 1988).

Although particles of 5 cm may pass through the reticulo-omasal orifice (Welch,1986), most particles leaving the rumen are smaller than 1 mm. Welch, 1986 cited that more than half (55.2%) of the ruminal DM passed through a .8-mm sieve. While mastication during feed ingestion and rumen fermentation play key roles in the diminution of particulate matter, rumination is the most important activity in reducing the particle size of indigested coarse material.

Selected stems from several different species and maturities of hay (2 cm of size) showed no change in physical form when incubated in ruminal fluid of fistulated steers in nylon bags for 10 d (Welch, 1982). Lengths of plastic ribbon 7-cm long with a .90 specific gravity required extensive rumination (particle size reduction) before they could pass from the rumen (Welch, 1982).

It is tempting to attribute size selection to the reticulo-omasal orifice. Mc Bride et al, cited by Welch, 1986, photographed the orifice using fiber optics and found that in feeder cattle the maximum opening was more than 4 mm, and more than 2 cm in cows (Welch, 1982); making it difficult to explain the separation solely on the basis of orifice action.

Since more than 50% of ruminal DM will pass a 600 μm sieve, other mechanisms, including particle density, and integrity of the ruminal mat are also likely to play important roles. Whatever the mechanism, it is clear that feed particles must be reduced to a size of less than 1 mm before they are likely to pass on to the lower digestive tract. The relationship between initial particle size of fiber (before being eaten) and ruminal retention time is given by the following equation: Kp = 3.21 - .016eNDF, where eNDF refers to the initial particle size of forage (before ingestion).


The Problem

Of primary concern with respect to fiber digestion is the consequent effect on energy intake and hence, animal performance. The rumen has an upper limit on its physical capacity. As the rate of fiber digestion decreases, the amount of slowly digestible OM in the rumen increases.

Zinn and Salinas (1999) observed that maximal DMI in cattle is a predictable function of initial weight (IW, kg) of cattle, dietary NDF (%), dietary eNDF (expressed as a percentage of dietary NDF), and ruminal NDF digestibility (PRDNDF, %): DMImax = (((.098 * IW) + 26.24) * (BW.75)) / ((.01 * NDF * (1 - (.01 * PRDNDF))) / ((.77 - (.00386 * ENDF)) * (-.037 + (.042 * NDF) - (.00031 * (NDF2))))). With a 220 kg calf fed a growing diet containing 21% NDF, a 20% reduction in ruminal fiber digestion is expected to depress the ADG by 10% (1.15 vs 1.28 kg/d).

With a 630 kg lactating cow fed a diet containing 30% NDF (80% eNDF), decreasing ruminal NDF digestion from 45% to 35% would drop maximum DM intake from 25 to 21 kg/d, resulting in a 21% decrease in milk yield (37 vs 47 kg/d).


Role for Enzyme Supplementation

Deficiencies in ruminal fibrolytic capacity may be partially overcome by enzyme supplementation. Combinations of cellulase and xylanase enzymes have enhanced in vitro (Feng et al., 1996; Howes et al., 1998) and in vivo NDF digestion (Lewis et al., 1996; Zinn and Salinas, 1999; Lopez-Soto et al., 2000; Murillo et al., 2000; Ware and Zinn, 2001), growth performance of steers fed forage-based diets (Beauchemin et al., 1995; Zinn and Salinas, 1999), and milk production (Howes et al., 1998). Listed below are abstracts of research we have conducted during the past 3 year involving fibrolytic enzyme supplementation.


INFLUENCE OF XYLANASE ON DIGESTIVE FUNCTION AND GROWTH PERFORMANCE OF FEEDLOT STEERS FED A 78% CONCENTRATE GROWING DIET

Zinn and Salinas (1999)

ABSTRACT: Eight Holstein steers (159 kg) with cannulas in the rumen and proximal duodenum (Zinn and Plascencia, 1993) were used in a crossover design to evaluate the influence of Fibrozyme supplementation (0 versus 15 g/steer/d) on digestion of a 22% forage, steamflaked corn-based growing diet. There were no treatment effects (P > .10) on ruminal pH, ruminal microbial efficiency, and ruminal and total tract digestion of OM and starch.

Fibrozyme supplementation increased (P < .05) ruminal digestion of NDF (23%) and feed N (5%). The increase in ruminal degradation of feed N is consistent with the associative effects of fiber on accessibility of forage protein to the proteolytic process. Tract digestion of NDF and N were similar (P > .10) across treatments. Ninety-six crossbred steer calves were used in a 64-d receiving trial to evaluate the influence of Fibrozyme supplementation on growth performance.

The trial was initiated July 21, 1998. Calves were blocked by weight and assigned within weight groupings to 16 pens (6 steers/pen). Upon initiation of the trial steers were implanted with Synovex-S (Fort Dodge Animal Health, Overland Park, KS).

Treatments were the same as in the metabolism trial. Calves were allowed ad libitum access to experimental diets. Fresh feed was provided twice daily (roughly 0700 and 1500 hr). Fibrozyme (7.5 g/steer, twice daily) was topdressed on the feed at the time of feeding. Fibrozyme supplementation increased final weight (3% , P < .10), ADG (6%, P = .13), and DM intake (4.5%, P < .05). Consistent with the metabolism trial, Fibrozyme did not influence the NE value of the diet. Treatment effects on DM intake (and consequently weight gain), can be explained by changes in ruminal NDF digestion.

The rumen has an upper limit on its capacity. As energy density of the diet decreases, the amount of slowly digestible OM in the rumen increases. In situations where energy density of the diet is limiting DM intake, maximal DM intake (DMImax) can be explained on the basis of effective NDF (eNDF) intake and ruminal NDF digestion. Accordingly, the 23% increase in ruminal NDF digestion due to Fibrozyme supplementation is expected to permit a 12% increase in DM intake.

Because the forage level of the basal diet was only marginally limiting energy intake, the observed increase in DM intake (4.5%) with fibrozyme supplementation was less then projected.

We conclude that supplementation of high-energy growing diet for feedlot cattle with Fibrozyme can enhance ruminal fiber digestion, and thereby may enhance dry matter intake and growth performance.


INTERACTION OF MACERATION AND FIBROLYTIC ENZYME SUPPLEMENTATION ON THE SITE AND EXTENT OF DIGESTION IN RICE STRAW IN HOLSTEIN COWS

Lopez-Soto et al (2000)

ABSTRACT: Five Holstein cows (633 kg) with cannulas in the rumen and proximal duodenum were used to evaluate the interaction of maceration and fibrolytic enzyme supplementation on digestion of rice straw. Treatments consisted of a steam-flaked corn-based diet supplemented with 40% forage as: 1) ground sudangrass hay; 2) ground rice straw, 3) macerated rice straw; 4) ground rice straw plus Fibrozyme (15 g/d), and 5) macerated rice straw plus Fibrozyme.

Enzyme supplementation increased ruminal digestion of OM (5%; P < .01) and NDF (21.8%; P < .01), but did not affect (P > .10) total tract digestion of OM, NDF, or N. Maceration decreased ruminal digestion of NDF (14%, P < .10), but increased (10%, P < .05) total tract NDF digestion. There were no treatment effects (P > .10) on ruminal microbial efficiency. Maceration decreased both ruminal (16%, P < .05) and total tract (4%, P < .01) N digestion. Maceration increased (P < .01) the Kp and Kd of NDF by 52% and 26%, respectively. Accordingly, maceration decreased ruminal solids and NDF content (13, and 28%, respectively; P < .10).

Enzyme supplementation increased the Kd of NDF (43%, P < .01). The increase in rate of NDF digestion with enzyme supplementation was greater (interaction, P < .05) with macerated than with nonmacerated straw (64 vs 19%, respectively). Enzyme supplementation did not affect (P > .10) ruminal solids and NDF content, or Kp of NDF. Total tract digestion of OM, NDF, and GE were greater (3, 19, and 4%, respectively; P < .05) for sudangrass- than for rice straw-based diets. There were no treatment effects on ruminal pH, VFA and estimated methane production. We conclude that enzyme supplementation works synergistically with maceration to enhance the feeding value of low quality forages.


INTERACTION OF FORAGE LEVEL AND FIBROLYTIC ENZYMES ON DIGESTIVE FUNCTION IN CATTLE

Murillo et al.(2000)

ABSTRACT: Four Simmental bulls (550 kg) with cannulas in the rumen and duodenum were used in a 4 × 4 Latin square design to evaluate the interaction of forage level ( 66 vs 33 %) and fibrolytic enzymes (0 vs 15 g/d Fibrozyme) on characteristics of digestion. Chromic oxide (0.19 %; DM basis) was added as a digesta marker. Fibrozyme (7.5 g/feeding) was incorporated into the diet at the time of feeding. Feed intake was restricted to 2.2 % of BW.

Increasing forage level increased (6%, P < .01) ruminal pH. There were forage level by enzyme interactions on ruminal digestion of the OM ( P _ .05 ), NDF ( P _ .05 ), ADF ( P _ .01 ), and starch ( P _ .10 ).

With diets containing 33% forage enzyme supplementation increased ruminal NDF and ADF digestion (10 and 43%, respectively). Whereas, with the diet containing 66% forage, enzyme supplementation did not affect fiber digestion.

Increasing forage level decreased ruminal microbial efficiency (11%, P < .01), and postruminal digestion of OM (13%; P < .01), NDF (36%; P < .01) , ADF (39%; P < .05), and starch (3%; P < .05). There were interactions (P < .01) between supplemental enzymes and forage level on total tract ADF digestion. As with ruminal digestion, enzyme supplementation also increased (8%) total tract ADF digestion in bulls fed the 33% forage diet, but it did not affect ADF digestion in bulls fed the 66% forage diet.

Increasing forage level decreased (3%, P < .01) total tract OM digestion and increased (8%, P < .01) total tract NDF digestion. We conclude that the responses to supplemental fibrolytic enzymes are augmented under conditions of lower ruminal pH.


INTERACTION OF DIETARY eNDF LEVEL AND SUPPLEMENTAL FIBROLYTIC ENZYMES ON SITE AND EXTENT OF FIBER DIGESTION IN CATTLE FED A 35% FORAGE GROWING DIET

Ambrozio, et al. ( 2001)

ABSTRACT: Eight Holstein steers (250 kg) with cannulas in the rumen and proximal duodenum were used in a replicated 4 × 4 Latin square experiment to evaluate the interaction of dietary eNDF (53, 65, 78, and 90% eNDF) and Fibrozyme® (an enzyme blend having both xylanase and cellulase activity; Alltech., Nicholasville, KY) supplementation on digestive function. Fibrozyme was added to the diet at time of feeding. Level of eNDF as a percentage of total dietary NDF did not influence (P > . 10) either ruminal microbial efficiency or site and extent of OM, NDF, starch and N digestion.

Fibrozyme supplementation also did not influence (P > .10) starch digestion, but it increased ruminal digestion of NDF (42.5 vs 50.3%, P < .01) and feed N (11%, P < .10). Fibrozyme did not influence (P > .10) total tract NDF digestion. We conclude that when ruminal NDF digestion is less than 45%, supplementation with fibrolytic enzymes will enhance ruminal NDF digestion. Under conditions where digestive tract fill is a limiting factor on energy intake, improving ruminal NDF digestion may enhance energy intake and hence, animal performance.


INFLUENCE OF FIBROZYME ON GROWTH PERFORMANCE OF YEARLING STEERS

Pereira and Zinn (2001)

ABSTRACT: Seventy-two yearling crossbred steers (348kg) were used in 121-d randomized complete block experiment to evaluate the influence of Fibrozyme® (an enzyme blend having both xylanase and cellulase activity; Alltech Inc., Nicholasville, KY; 0 vs 15g/d) supplementation on growth performance.

During the first 84-d (growing phase) the basal diet contained 22% forage and 65% steam-flaked sorghum. From d-85 to d-121 (finishing phase) the basal diet contained 12% forage and 75% steam-flaked sorghum. Fibrozyme® supplementation increased (P < .10) ADG 6% during the growing phase, and 20% (P < .05) during the finishing phase.

Over the entire study, Fibrozyme® supplementation increased ADG 10% (P < .01). Fibrozyme® supplementation did not influence (P > .10) DMI. However, it did increase (P < .10) gain efficiency (6.3%) and dietary NEg (5.1%). Fibrozyme® supplementation increased (2.7%, P < .05) carcass weight, but did not influence (P > .10) dressing percentage.

Increased ADG without concurrent increases in DMI with Fibrozyme® supplementation were not expected, particularly during the late finishing phase when dietary fiber levels were low. Results suggest that Fibrozyme supplementation may enhance cattle performance in a manner independent of its effects on fiber digestion.


ENZYME SUPPLEMENTATION TO OVERCOME THE NEGATIVE ASSOCIATIVE EFFECTS OF SUPPLEMENTAL FAT ON FIBER DIGESTION IN CATTLE

Ware and Zinn (2001)

ABSTRACT: Four Holstein steers (522 kg) with cannulas in the rumen and duodenum were used in a 4 × 4 Latin square design to evaluate the interaction of supplemental fat ( 0 vs 4%) and fibrolytic enzymes (0 vs 15 g/d Fibrozyme) on characteristics of digestion. Fibrozyme was incorporated into the diet at the time of feeding. Feed intake was restricted to 2% of B W.

Fat supplementation depressed ruminal digestion of OM (14%, P < .01) and N (10%, P < .05). There was a supplemental fat by enzyme interaction (P < .10) on ruminal NDF digestion. In the absence of supplemental fat, ruminal NDF digestion was high (51 %) and not affected by enzyme supplementation. In the absence of supplemental enzyme, fat supplementation depressed ruminal NDF digestion (30%, P < .05). Fibrozyme addition to the fat supplemented diet increased (25%, P < .05) ruminal NDF digestion to a level similar to that of non-fat supplemented diets (47%).

Fibrozyme supplementation tended (6%, P < .10) to increase ruminal degradation of feed N. There were no treatment effects (P = .45) on ruminal microbial efficiency. There were no treatment interactions (P > .10) on total tract digestion. Fat supplementation depressed total tract digestion of OM (5%, P < .O1), NDF (17%, P < .O1), and N (4%, P < .05). Fibrozyme supplementation increased (8%, P < .10) total tract digestion of NDF. We conclude that Fibrozyme supplementation can overcome the negative associative effects of supplemental fat on ruminal fiber digestion of growing-finishing diets fed to feedlot cattle.


Literature Cited

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