Abstract:
A method for increasing the rate and extent of fiber digestion in a ruminant after ingestion by the ruminant is described. The method includes forming an aqueous mixture including carbohydrase enzymes active on structural carbohydrates of a forage and applying the aqueous mixture to the forage prior to ingestion by the ruminant at an application ratio grater than about 20 milliliters of the aqueous mixture per pound of forage dry matter.

Description:
This patent application claims the benefit of provisional patent application Ser. No. 60/021,186 which was filed on Jul. 3, 1996. 
    
    
     FIELD OF THE INVENTION 
     This invention relates generally to a method for increasing the digestibility of ruminant feed with exogenous enzymes, and in particular to a method of applying a dilute aqueous mixture of carbohydrase enzymes to ruminant feed at a high ratio of the aqueous mixture to feed so that the rate and extent of fiber digestion by the ruminant are increased. 
     BACKGROUND OF THE INVENTION 
     Prior research on the use of enzymes as feed additives has resulted in the creation and application of commercial products in the swine or poultry industry. In these applications, the applied enzymes supplement the endogenous digestive enzymes in the host animal; remove anti-nutritional factors such as β-glucans from problem feedstuffs such as barely; render certain nutrients readily available and enhance the energy value of feed stuffs. However, prior research on the addition of enzymes to ruminant diets has not supported the feeding of carbohydrase enzyme preparations to improve ruminal fiber digestion. This is due to the rapid degradation of the unprotected exogenous enzyme by ruminal bacterial protease which occurs prior to the enzyme effecting carbohydrate digestion. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to the application of carbohydrase enzymes to diets of ruminate animals to increase the rate and extent of fiber (primarily forage) digestion by the ruminate. The enzymes are introduced into the diets by application of a dilute aqueous mixture of carbohydrase enzymes to the forage prior to ingestion by the animal. The present invention is also directed at enzyme application to diets of ruminant animals fed in commercial operations. 
     In one embodiment of the invention there is provided a method for increasing the rate and extent of fiber digestion in a ruminant, including forming an aqueous mixture of carbohydrase enzymes active on structural carbohydrates of a forage and applying the aqueous mixture to the forage prior to ingestion by the ruminant at an application ratio greater than about 20 milliliters of the aqueous mixture per pound of forage dry matter. 
     It is understood with respect to digestion, the term &#34;rate&#34; indicates the relative speed at which the fiber is broken down and &#34;extent&#34; corresponds to the total amount of fiber digested. 
     The novel aspects of this invention are set forth with particularity in the appended claims. The invention itself, together with further objects and advantages thereof, may be more fully comprehended by reference to the following detailed description of a presently preferred embodiment of the invention. 
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In addition to enzyme formulation, it has been discovered that the dilution of the enzymes and high application ratios of the aqueous moisture to forage are critical parameters in the overall effectiveness of enzyme preparations added to ruminant diets. It has been further discovered that the successful application of the enzymes to the fiber is dependent upon adequate dispersion of the enzyme(s) over the surface area of the forage particles, and is substantially independent of the enzyme formulation. It is believed that successful application requires the enzymes to be applied in a dilute aqueous solution wherein the solution is applied at a relatively high application ratio. For example, for a given recommended amount of enzyme concentrate per pound of forage dry matter, the enzyme concentrate is diluted so as to apply at least 20 ml and preferably 200 ml or more of the dilute aqueous solution per pound of forage dry matter (DM). With the relatively high volumes of dilute solution per pound, i.e. &gt;=200 ml, adequate dispersion can be obtained by spraying the solution over the feed load at a rate of approximately 10 to 40 gallons per minute while mixing for a relatively short time, approximately 3 to 5 minutes. 
     Relatively low volumes of solution per pound of forage dry matter (on the order of 20 ml dilute solution per pound of forage DM) have been found to yield beneficial results, however, the application time must be increased. That is, for these lower volumes of solutions, an application rate of approximately 0.5 to 2 gallons per minute is employed with an application time on the order of 10 minutes to achieve adequate dispersion of the enzymes. Generally, as the solution volume per pound of forage dry matter decreases, the length of application time increases to ensure that the enzymes are evenly distributed throughout the forage. 
     The aqueous mixture can be applied to ingestible materials, such as hay or other forage, immediately prior to ingestion. Preferably, the forage is mechanically turned as the spray is applied, for example by a reel or an auger, to ensure uniform coverage. The ingestible material is sprayed with the aqueous enzyme mixture from an applicator tube or a spray nozzle. In some applications, it may be desirable to heat the ingestible material prior to spraying. Alternatively, the aqueous diluents are heated to a maximum temperature of about 150° F. and mixed with the enzyme concentrate. The maximum application temperature is selected to substantially preclude degradation of the enzymes from thermal effects. Application of the diluted enzyme mixture at elevated temperatures may increase binding of the enzymes to the fiber. 
     For any given application rate, a spray nozzle configuration is selected to give adequate dispersion over the feed. It is preferred that the nozzle have a solid cone spray pattern to provide uniform distribution of the aqueous mixture to the feed. Suitable nozzles have an orifice size of from about 0.05 to 0.20 inches and a typical installation would have from two to six nozzles. The orifice size and number of nozzles selected depend on the rate of application required and the quantity of forage to be treated. Such methods of application are suitable for treating commercial quantities of forage, for example 500 LB to 3,000 LB of forage. 
     The diluent is usually water but other aqueous products or by-products can be used. Lacto-whey, a by-product of milk production which is predominantly water, for example 90% water, can be used as the diluent for the enzymes. 
     With the present application ratio of the aqueous mixture, the rate and extent of fiber digestion are increased. This increase may be obtained over a range of application temperatures at ambient or atmospheric pressure. That is, the present application ratio causes increased rate and extent of fiber digestion without requiring compression of the forage. Once coverage with the dilute solution is achieved, it is not necessary to subject the wetted forage to mechanical compacting pressure or compression after application and before feeding. Further, with the present application ratio, the dilute aqueous solution may be applied over a broad temperature range. In fact, it is believed the dilute solution may be applied from approximately a freezing temperature to 150° F. The upper temperature limit is substantially determined by the denaturing temperatures of the enzymes. 
     It is not necessary to compact or cube the feed before feeding. The process of cubing the feed requires exposing the feed to high humidity, elevated temperatures and pressure which may initiate and cause substantial fiber degradation by the enzymes prior to ingestion by the ruminant. 
     Specifically, the present invention is directed at the effects of enzymes active on structural carbohydrates (fiber) on the digestion of forage. These enzymes include, but are not limited to, carbohydrase enzymes active on a structural carbohydrate, for example, cellulase, cellobiase, arabanase, pectanase, polygalacturonase and xylanase. 
     It is also recognized that genetic engineering is creating previously unavailable carbohydrase enzymes, enzyme activity combinations and enzyme activity ratios which can be used for fiber degradation. Such engineering has produced specialized enzyme activities within a generic classification. For example, exocellulase and endocellulase now exist within the cellulase class of carbohydrase enzymes. 
     The enzymes may be commercially available in a concentrated form containing at least about 70% of active enzymes. The concentrate is preferably diluted with an aqueous diluent in a ratio of approximately 6 to 60 ml of the concentrate to approximately 400 to 16,000 ml of the aqueous mixture. In the present commercially available form, the concentrate is approximately 90% active enzymes and 10% carriers, such as glucose and glycerol. A suitable commercial carbohydrase enzyme concentrate, Rumenase, is available from Agriscience Inc. of Liverpool, N.Y. Rumenase concentrate typically contains four major enzymes with the following activities: 
     
         ______________________________________Cellulase          1,050        ECU/mlCellobiase         17.2         CBU/mlXylanase           43,000       BXU/mlPolygalacturonase  1,245        PGU/ml______________________________________ 
    
     At the preferred dilution, on a per head per day basis and assuming an intake of 20 lb. of forage dry matter per day, approximately 20 ml of the carbohydrase concentrate is added to 3,980 ml of water to form a 4,000 ml dilute aqueous solution, i.e. 200 ml of an aqueous mixture of carbohydrase enzymes per pound of forage dry matter. 
    
    
     EXAMPLES 
     In testing with lactating dairy animals, dietary forage of a barley based commercial concentrate, TMR (50% hay crop silage, 5% alfalfa hay and 45% barley-based concentrate) is sprayed with one of two treatments while mixing in a farm scale mixer wagon. The control group dietary forage is sprayed with water only. The treatment group dietary forage receives a dilute aqueous enzyme solution diluted as follows: 1.1 liter of enzyme concentrate (Rumenase) in about 250 liters of water per ton of forage dry matter. 
     The water and the aqueous enzyme mixture are applied with a hose end sprayer. 
     Sufficient time, at least about 3 minutes, is allowed for the enzyme to be absorbed and bound by the forage before it is fed to the cow. 
     The cow is given access to the forage for up to about 6 hours after treatment of the forage and is preferably fed twice per day. 
     TMR samples are evaluated in situ in their original wet form as follows: Approximately 5 g of material (dry weight) are weighed into forage bags and placed in the rumen of a fistulated lactating cow. Each set of samples are evaluated in the same cow on the same day to allow for comparison between treatments. 
     Samples are incubated for 12 or 24 hours in the rumen of the lactating dairy cow. 
     All in situ bags of TMR samples for a given time point are placed into a weighted nylon mesh bag and attached to the cover of the ruminal cannula with a length of nylon cord sufficient to allow free movement in the rumen. 
     A single nylon mesh bag is removed at the end of each incubation period. Immediately after removal from the rumen, samples are immersed in cold (11° C.) water to end fermentation and remove extraneous ruminal contents. In situ bags are subsequently removed from the nylon mesh bag and rinsed three times in three volumes of cold water. Samples are frozen until analysis is performed. The samples are thawed out, washed with cold water, dried at 60° C. in a convection oven for 48 hours and then weighed. Dry matter disappearance is calculated. Neutral detergent fiber (NDF) disappearance is determined by conventional wet chemistry procedures for both original material and in situ residues. 
     In the following Examples 1-3 the forage was treated with water or dilute Rumenase at an application ratio of 250 ml/LB forage dry matter. The diet was fed to cows so that each cow received 20 LB dry forage matter diluted with approximately 5 liters of water or aqueous enzyme mixture. Access to the feed was limited to a maximum of 6 hours. 
     Example 1 
     Digestibility studies on individual forage ingredients and on TMR&#39;s treated with water and aqueous Rumenase. 
     
         ______________________________________NDF/DIGESTIBILITIESTREATMENT  Corn Silage  Alfalfa Hay                             TMR______________________________________Water      20.1%        25.0%     23.2%Rumenase   35.2%        34.4%     31.8%______________________________________ 
    
     Temperature effect: A neutral detergent fiber (NDF) was treated with water at 40° F., diluted Rumenase at 40° F. or diluted Rumenase at 120° F. and the level at digestibility of the feed was determined. 
     
         ______________________________________TREATMENT    Temp (°F.)                  NDF/DIGESTIBILITY______________________________________Untreated    40        12.7%Rumenase     40        16.0%Rumenase     120       21.7%______________________________________ 
    
     Example 3 
     Diluent effect: Samples of NDF were treated with either whey or Lacto-whey. Lacto-whey is regular whey that is ammoniated with ammonia to raise the protein content. Rumenase diluted with water or Lacto-whey was sprayed on the feed and fed to cows. 
     
         ______________________________________     DILUENT NDF/DIGESTIBILITY______________________________________TRIAL 1Untreated             26.4%Rumenase    water     50.7%Rumenase    whey      48.2%TRIAL 2Untreated             21.9%Rumenase    water     33.6%Rumenase    Lacto-whey                 30.1%______________________________________ 
    
     Example 4 
     Samples of corn silage were treated with Rumenase diluted at a low dilution rate (20 ml/LB of forage dry matter) and a high dilution rate (200 ml/LB of forage dry matter). At both dilution rates the level of Rumenase was constant at 0.75 ml/LB of forage dry matter. 
     
         ______________________________________NDF/DIGESTIBILITY (%)            12 hour                  24 hour______________________________________Control            6.9     18.4Rumenase (low dilution)              9.4     17.9Rumenase (high dilution)              12.6    20.0______________________________________ 
    
     The results of this trial showed that the addition of Rumenase at the low dilution rate increased the rate of digestion, but not extent of digestion. This is indicated by the 36% increase in NDF disappearance at 12 hours, but no improvement at 24 hours. However, the addition of Rumenase with a high dilution rate increases NDF disappearance at both 12 and 24 hours, 83% and 9% respectively, thereby enhancing the extent of digestion. 
     The present method increases the rate and extent of fiber digestion within the ruminant without significant initiation of enzymatic action prior to ingestion so that essentially all of the enzymatic action is in the ruminant. 
     While the invention has been described in connection with a presently preferred embodiment thereof, those skilled in the art will recognize that many modifications and changes may be made therein without departing from the true spirit and scope of the invention which, accordingly, is intended to be defined solely by the appended claims.