Method of maintaining ruminants on high energy low fiber diet
FIELD OF THE INVENTION 
     This invention relates to the use of parasympathomimetic compounds, their precursors, salts and metabolites, to alter the digestive process in livestock so as to increase the efficiency of food utilization while simultaneously reducing the risk of certain disorders frequently associated with high energy diets. Specifically, the invention comprises administration to ruminants and other livestock of low level dosages of a parasympathomimetic compound to increase salivation during feeding and rumination, thereby increasing rumen digesta turnover rates and efficiency of food utilization while reducing the risk of certain digestive tract disorders such as acidosis and displaced abomasum. 
     BACKGROUND OF THE INVENTION 
     Rapid production systems currently employed with respect to cattle, sheep and other domestic ruminants typically entail intensive use of relatively expensive high energy feedstuffs to maximize growth rates. Examples include any feedstuffs having a high starch content or a high percentage of total digestive nutrients (TDN). Usually, these feedstuffs contain relatively large quantities of grain or other finely ground energy and protein sources. Although these feedstuffs are almost always high in potential energy content, they differ substantially in fiber content from the grasses, roughage and other forages which it is generally believed ruminants evolved to consume. 
     As a result, stresses are created in the digestive tracts of ruminants which are maintained on relatively high energy, low fiber diets. The nature and collective age of the microbial population which exists in the rumen in a symbiotic relationship with the animal can be detrimentally altered, leading to increased production of volatile fatty acids and a subsequent increase in systemic acid load. In severe cases, these stresses can lead to acute digestive dysfunctions, such as acidosis, displaced abomasum and other pathological conditions. However, even in mild, subclinical cases, these stresses decrease the efficiencies with which ruminants digest and utilize high energy feedstuffs. Ultimately, inefficiencies are created in the overall production system, and they are compounded by both the expense of underutilized, high energy or high protein feedstuffs and the cost where veterinary or other corrective treatment is required. 
     Insufficient salivation, brought about by the relatively minimal chewing necessary to consume fine particulate, high energy feedstuffs has previously been recognized as a primary cause of many of these disorders. It has been reported that saliva is the source of up to 70% of the total water flux through the rumen as well as the principal source of ruminal buffering agents. Additionally, it is known that high energy diets promote increased production of volatile fatty acids. Thus, animal scientists have recognized that a significant decrease in ruminant salivation can lead to excess acidity, decreased digesta flow to the small intestine, a slower rate at which dietary nutrients are washed out of the rumen into the small intestine and an alteration in the nature and collective age of the microbial population which exists in the rumen in symbiosis with the host animal. 
     Heretofore, practical efforts to address the problems arising from feeding ruminants a high energy diet have tended toward treatment of symptoms rather than finding cures or true preventatives. In large part, these efforts have focused upon increasing the fiber content of feedstuffs or upon supplementing high energy diets with salts, minerals or buffering agents such as sodium chloride, calcium chloride, limestone, various bicarbonates and bentonites. While these measures have been of some assistance in enhancing rumen digesta turnover and in controlling excess acidity as well as some of the other digestive dysfunctions associated with high grain diets, they have been substantially less than completely successful. 
     It is known that, by stimulating the exocrine glands, certain parasympathomimetic compounds significantly enhance salivation and pancreatic fluid secretion in ruminants and other animals. However, these compounds have been subject to a lack of reliable information and predictability regarding the amount of salivation they induce and as to whether they can be administered repeatedly to animals over a prolonged period. Additionally, these compounds have been reported to produce other undesirable effects, such as diarrhea, increased urination and decreased heart and respiratory rates. Some of these compounds have been reported to be toxic and have been the subject of significant efforts to purify certain feedstuffs. Thus, prior to this invention, parasympathomimetic compounds had not been adopted for maintaining any animals on any diet. 
     Slaframine, one of the naturally occurring indolizine alkaloids produced by the fungus Rhizoctonia leguminicola, is a parasympathomimetic compound which is known to cause increased salivation. In the Midwest and elsewhere in the 1950&#39;s, 1960&#39;s and 1970&#39;s, there were periodic outbreaks among cattle and other livestock of salivary syndrome or &#34;slobbers&#34;. Typically, the livestock showed pathologic symptoms such as excessive salivation, decreased feed intake, abortion, diarrhea, lacrimation and behavioral changes. In some instances, the livestock died. 
     &#34;Slobbers&#34; was traced to consumption by the livestock of red clover hay containing the fungus, Rhizoctonia leguminicola. Slaframine, was reported to be the causative factor and was subsequently reported to be a toxin. More recently, however, it has been discovered that swainsonine (1,2,8-trihydroxyoctahydroindolizine), another compound co-produced with slaframine by Rhizoctonia leguminicola, is the active toxin produced by that fungus. 
     We have discovered that low level dosages of purified slaframine (i.e., slaframine in the absence of significant quantities of swainsonine) can be administered to cattle and other ruminants over prolonged periods of time so as to increase rumen digesta turnover rates and alter a variety of other rumen parameters in ways which are quite beneficial to livestock maintained on relatively high energy, low fiber diets. Moreover, we have discovered that, when administered to ruminants in accordance with this invention, slaframine does not produce pathological conditions. 
     SUMMARY OF THE INVENTION 
     A primary object of this invention is to provide a method for maintaining ruminants on a relatively high grain, low fiber diet while reducing the risks of acidosis, displaced abomasum and other digestive dysfunctions. 
     Another object of this invention is to provide a practical method, through the use of low level dosages of parasympathomimetic compounds, for increasing salivation of ruminants and to do so in a controlled manner, without debilitating side effects. 
     Another object of this invention is to provide a method for increasing digesta flow from the rumen, thus increasing the rate at which nutrients are washed out of the rumen into the lower digestive tract, thereby increasing the efficiency of high energy feedstuff utilization by ruminants. 
     Still another object of this invention is to regulate the extent and rate of microbial growth in the rumen by controlling pH of rumen digesta and rumen digesta flow. 
     We have found that, when low level dosages of purified parasympathomimetic compounds are administered to ruminants at about the time of feeding, they can be maintained indefinitely on high energy, low fiber diets without suffering digestive tract disorders. Depending upon its specific identity and state at the time of administration (i.e., whether in free base, salt or other forms), the parasympathomimetic compound can be administered intramuscularly, intraperitoneally, intravenously, subcutaneously or orally. By stimulating the exocrine glands and by increasing salivation on a significant scale but in a controlled manner, these compounds decrease the risk of rumen dysfunction. Additionally, they increase water flux through the rumen, and rumen digesta turnover rates, thereby decreasing the risk of displaced abomasum. Furthermore, an exciting finding is that slaframine alters the rumen environment in such a manner as to promote more efficient use of relatively costly high energy feedstuffs. 
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     A variety of parasympathomimetic compounds, such as pilocarpine and carbacol appear suitable for use in accord with this invention. However, the preferred compound is slaframine (1-acetoxy-6-aminooctahydroindolizine), one of several indolizine alkaloids produced by the asporogenous fungus Rhizoctonia leguminicola. 
     The compound is administered in an amount insufficient to produce a refractory condition (such that the animal fails to respond to various stimuli) but sufficient to increase salivation and the digesta flow from the rumen. Generally, a dosage range of about 12-24 micrograms per kilogram, body weight, injected intramuscularly once every 8-12 hours has been found satisfactory, with 15-20 micrograms per kilogram, body weight, once every 8-12 hours, being the optimum dosage range in cattle. In most ruminants, this dosage may be repeated at regular intervals, or administered as a continuous subcutaneous implant or inunction with recurrent effectiveness and without undesirable effects on feed intake, heart rate, respiratory rate or kidney output. 
     The effects on various ruminal parameters of red clover containing 7 micrograms per gram slaframine when fed to four sheep consuming a pelleted, high energy diet is shown in Table I. Addition of slaframine altered the rumen environment and increased the acetate/propionate ratio by 27-44%, rumen fluid turnover rate by 35-38% and salivary flow by 24-36%. 
     
                       TABLE I                                                     
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Effects of Daily Feeding of Various Amounts                               
of Red Clover Hay Continuing Slaframine on                                
Ruminal Acetate/Propionate, Fluid Turnover                                
Rate, Volume and Salivary Flow in Wethers                                 
Fed a Pelleted Grain Diet                                                 
             Red Clover Hay Fed (g/day).sup.a                             
Item           Control.sup.b                                              
                        25       50   100                                 
______________________________________                                    
Acetate/Propionate                                                        
               2.14     2.18     2.72 3.08                                
Rumen fluid turnover                                                      
               5.28     6.48     7.30 7.12                                
rate (% hour)                                                             
Salivary flow (ml/24 hr)                                                  
               2534     2258     3450 3142                                
______________________________________                                    
 .sup.a Red clover hay contained 7 ug of slaframine.                      
 .sup.b Control = 50 g of ground alfalfa pellets daily.                   
 
    
     The effects of slaframine administered intramuscularly on resting salivary flow rate in Angus steers is shown in Table II. Results confirm data obtained from withers (Table I) and demonstrate the practicality of obtaining controlled increases in salivary flow in steers. In steers, resting salivary flow accounts for a minimum of 50-80% of total flow. The steers were fed a high energy diet at about twice maintenance--roughly equivalent to feed lot levels. 
     
                       TABLE II                                                    
______________________________________                                    
Effect of Intramuscular Injection of Graded Doses                         
of Pure Slaframine on Resting Salivary Flow in                            
Angus Steers                                                              
       Time in relationship to dosing (hr)                                
Dosage   -2 to -1  +.5 to 3.5                                             
                             +4.5 to 6.5                                  
                                     +7.5 to 8.5                          
(ug/kg BW)                                                                
         ml/min                                                           
______________________________________                                    
 0       37.8      44.8      36.1    39.2                                 
 6       30.7      42.7      37.0    36.2                                 
12       37.1      57.1      49.7    40.2                                 
24       34.7      62.8      53.6    43.2                                 
Significance                                                              
         NS        .01       .01     NS                                   
______________________________________                                    
 Steers fed high energy, low fiber diet 24 × daily at twice         
 maintenance levels.                                                      
 
    
     No undesirable effect on feed intake, heart rate, respiratory rate or kidney output was observed. 
     Pure slaframine is a colorless oil which is unstable on exposure to air. It is, however, stable as the free base in such as chloroform and methylene chloride. Salts of slaframine, such as the picrate, dipricate, citrate, hydrochloride, sulfate and tartrate salts are generally more stable; and most of them are suitable for prolonged storage. The dipricrate may be the most stable, but the free base must be regenerated and separated from the picric acid before the drug can be administered to animals. The other salts are ideal for storage and preparations can be administered directly to animals either orally or by injection in buffered saline. 
     The chemical structure of slaframine is as follows: ##STR1## Slaframine must be activated in the animal&#39;s body, apparently in the liver, to exert its parasympathomimetic effects. This phenomenon is also of great benefit in achieving sustained release of the active metabolite and extending the duration of effectiveness of the administered drug. Because one mole of ammonia is liberated per mole of slaframine during activation, a deamination yielding the compounds A and B, below, is the metabolic mechanism leading to the metabolite of slaframine which has been reported to be the active parasympathomimetic agent, 1-keto-6-aminooctahydroindolizine, (C 8  H 15  N 2  O), compound B, below: ##STR2## 1,6-dihydroxyoctahydroindolizine 1-keto-6-aminooctahydroindolizine See Aust, Rhizoctonia leguminicola--Slaframine, pp. 97-109 in Mycotoxins, edited by I.F.H. Purchase (Elsevier Scientific Publishing Company, N.Y. 1974). 
     The biosynthetic pathway through which R. leguminicola generates slaframine has been shown to include the cyclization of lysine to pipecolic acid then further biochemical modifications to form the apparently nonparasympathomimetic 1-hydroxy-6-aminooctahydroindolizine which is then acetylated to yield the strongly active slaframine. See Aust, S. D. 1965 Doctoral Thesis, University of Illinois; Guengerich, &#34;Lysine and Slaframine Biogenesis,&#34;  30 Fed. Proc. 1067 (1971). Inclusion of serine, lysine, pipecolic acid into the growth medium for R. leguminicola has been shown to stimulate the production of slaframine, which, however, is not secreted into the growth medium but retained in the mycelium. This greatly facilitates isolation and purification because extraction of large amounts of liquid culture filtrate has been shown to be unnecessary. Yields of 3-5 mg/g mycelial dry weight have been achieved with slaframine accumulation paralleling growth. Swainsonine may be separated from slaframine by a countercurrent extraction using methylene chloride and partitioning against water at a pH of 10. 
     Two publications in the early 1970&#39;s reported successful chemical synthesis of slaframine. Cartwright, Gardner and Rinehart, &#34;Synthesis of Slaframine,&#34; 92 Journal of the American Chemical Society 7615 (1970); Gensler and Hu, &#34;Synthesis of D, L-Slaframine,&#34; 38 Journal of Organic Chemistry 3848 (1973). However, routes to chemical synthesis are presently less efficient than slaframine production by fermentation. There is considerable information on biological production of slaframine by fermentation as well as the chemistry of slaframine so that the potential for large-scale production of this alkaloid drug is great. The potential for strain modification to increase efficiency of fermentations through biotechnology is also great, given the low costs and high yields presently obtained using systems developed for other natural fungal products such as penicillin or zeranol (Ralgro).