EXPERIMENTAL AMMONIA TOXICOSIS 1N HOLSTElN-FRlESlAN STEERS THS Thesis for the Degree of M. S. MICHIGAN STATE UNNERSITY DONALD A. HENSHAW 1969 LIBRARY ' “Ev“ Michigan. 3mm University . ._ — amnmo av ' , HMS 8: WHY I i WEEABEEBUEE- » ABSTRACT EXPERIMENTAL AMMONIA TOXICOSIS IN HOLSTEIN-FRIESIAN STEERS by Donald A. Henshaw Toxicosis due to the oral ingestion of urea was determined in approximately 375 kg. Holstein-Friesian steers. Toxicosis from selected ammonium compounds injected intravenously was also studied. Signs associated with overconsumption of urea were atony of the rumen, poly- uria, muscle tremors (ceased temporarily following urination), ataxia, tachycardia, extension of the legs, increased respiration, apparent blindness, paralysis, chronic convulsions, and death. The central nervous signs were more pronounced than with cyanide or nitrite pois- oning. Diagnosis and clinical signs were associated with increased levels of blood plasma ammonia nitrogen. Intravenous injections of ammonium carbonate, ammonium chloride, and ammonium oxalate caused clinical signs similar to those of urea toxicosis. Rumen atony occurred within 5 to 10 minutes after injection of ammonium compounds. When blood ammonia nitrogen levels increased to 0.6 mg./lOO ml. external clinical signs were evident and death occurred when levels were between 1.4 and 3.2 mg./100 ml. On necropsy, no characteristic gross or microscopic lesions were observed in a steer that died from intravenous injection of ammonium compounds. Marginal perivascular and perineuronal edema in all sections of the central nervous system, vacuolation of the hepatic cells of the kidney, diffuse Donald A. Henshaw hemorrhages on the mucosal surface of the abomasum and ecchymotic hemor- rhages in the endocardium and myocardium were present on necropsy of the steer that died from urea toxicosis. Blood urea nitrogen levels were an unreliable indication of urea toxicosis. This research indicated that the ammonium ion caused the toxic syndrome known as urea toxicosis. The testing procedures for blood plasma ammonia nitrogen levels used in this research could be run in any laboratory and would be a diagnostic aid in suspected cases of urea toxicosis. Additional determinations included blood pH and serum electrolyte levels. The results indicated that ammonium compounds injected intra- venously and urea administered orally had minimal effects on blood pH or serum electrolytes. EXPERIMENTAL AMMONIA TOXICOSIS IN HOLSTEIN-FRIESIAN STEERS By Donald A. Henshaw A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Veterinary Surgery and Medicine 1969 ACKNOWLEDGEMENTS The author wishes to express his appreciation to the guidance committee, Dr. G. H. Conner, Dr. D. J. Ellis, Dr. W. F. Riley, Jr., Dr. R. W. Van Pelt, and Dr. C. K. Whitehair, for their guidance and counsel. Special thanks goes to Dr. D. J. Ellis, major advisor, and to Dr. C. K. Whitehair for their assistance in this research project and to Dr. S. D. Sleight and Dr. G. H. Conner for their advice and criti- cal reading of this thesis. The author wishes to express his sincere appreciation to Miss Irene Brett, Mrs. Ruth Kelly, and Mrs. Virginia Chen for their help in some of the laboratory blood determinations. To his wife, Mary, for her encouragement and understanding, goes the author's undying gratitude. ii INTRODUCTION . . . . . . . REVIEW OF THE LITERATURE . Urea Metabolism . . Urea Toxicosis. . . TABLE OF CONTENTS Ammonia Toxicosis in Ruminants. Ammonia Metabolism in Man . . MATERIALS AND METHODS. . . Blood Plasma Ammonia Nitrogen . Blood Urea Nitrogen . Blood pH. . . . . . Serum Electrolytes. Magnesium. . Phosphorus . Calcium. . . Chloride . . Sodium and Potassium . Experimental Ammonia Toxicosis. Experimental Animals . Preparation of Experimental Ammonium Compounds . Ammonium Compound Injection Technique. . . . . Blood pH . . Serum Electrolytes . . Blood Urea Nitrogen. . iii Page 13 13 14 16 16 l7 17 17 17 18 18 18 18 20 20 20 20 Plasma Ammonia Urea Experiments. . . Necropsy. . . . . . . RESULTS. . . . . . . . . . . DISCUSSION . . . . . . . . . Additional Procedures SUMMARY. . . . . . . . . . . REFERENCES . . . . . . . . . VITA O O O O O O O O O O O O Nitrogen. iv Page 20 21 22 23 42 47 48 51 57 Table 10 ll 12 LIST OF TABLES Page Determination of average blood plasma ammonia nitrogen level in cattle O O O O O O O O O O O O O O O O O O O I O O 15 Identification and weight of experimental Holstein- Friesian steers, duration of treatments, ammonium treat- ments, dates of injection . . . . . . . . . . . . . . . . . 19 Weight of Holstein-Friesian steer No. 2, duration of treatment, urea levels, method of treatment, dates of treatment 0 O O O O O O O I O O O O O O O 0 O O O O O I O O 19 Effect of delayed reading time on blood plasma ammonia nitrogen levels in Jersey cattle. . . . . . . . . . . . . . 24 Effect of delayed reading time on blood plasma ammonia nitrogen levels 0 O O O O O O O O O O O O O O O O I O O O O 24 Effect of delayed reading time on blood plasma ammonia nitrogen levels 0 O O O O O O O O O O O O I O O O O O O O I 25 Effect of brome grass and orchard grass pasture on blood plasma ammonia nitrogen levels in Jersey cattle . . . . . . 26 Effect of brome grass and orchard grass pasture on blood pH Of Jersey cattle O O O O O O O O O O O O O O O O O O O 6 27 Effect of delayed reading on blood pH in Guernsey and Holstein-Friesian cattle. . . . . . . . . . . . . . . . . . 28 Effect of exposure to brome grass and orchard grass pasture on blood serum magnesium, phosphorus, calcium, chloride, sodium and potassium levels of Jersey cattle. . . 29 Effect of the intravenous injection of 500 milliliters of 4% ammonium carbonate, 6% ammonium carbonate, and 7% ammonium oxalate on the blood pH of Holstein-Friesian steers. . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Effect of the intravenous injection of 500 milliliters of 4% ammonium carbonate and 8% ammonium carbonate on the blood serum chloride, sodium, and potassium levels in Holstein-Friesian steer No. l. . . . . . . . . . . . . . 32 Table Page 13 Effect of the intravenous injection of 500 milliliters of 1.6% ammonium carbonate, 4% ammonium carbonate, and 8% ammonium carbonate on the blood urea nitrogen levels in Holstein-Friesian steer No. l . . . . . . . . . . . . . . . 33 14 Effect of the intravenous injection of 500 milliliters of 1.6% ammonium carbonate, 4% ammonium carbonate, 6% ammonium carbonate, 7% ammonium carbonate, 8% ammonium carbonate, 8% ammonium chloride and 7% ammonium oxalate on blood plasma ammonia nitrogen levels in Holstein-Friesian steers. . . . . . . . . . . . . . . . . . . . . . . . . . . 34 15 Effect of the administration of urea on the blood pH of Holstein-Friesian steer No. 2 . . . . . . . . . . . . . . . 35 16 Effect of urea on the blood urea nitrogen level of Holstein-Friesian steer No. 2 . . . . . . . . . . . . . . . 35 17 Effect of urea on the blood plasma ammonia nitrogen level in H01Stein-FrieSian Steer No 0 2 O O C O O O C O O O O O I O O 37 vi LIST OF FIGURES Figure Page 1 Holstein-Friesian steer No. 2 15 minutes after adminis- tration of 272 Gm. of urea. . . . . . . . . . . . . . . . . 38 2 Initial urea toxicosis, 30 minutes after urea administra- tion. 0 O O O O O O O O O O I O O O O O 0 O O O O O O O O O 39 3 Urea toxicosis. Increasing toxic signs 45 minutes after urea administration . . . . . . . . . . . . . . . . . . . . 40 4 Urea toxicosis. Paralysis prior to death. Sixty minutes after administration. . . . . . . . . . . . . . . . 41 vii MATERIALS AND METHODS During the course of the experiments, which will be outlined later, the following determinations were made: blood plasma ammonia nitrogen, blood urea nitrogen, blood pH and serum electrolytes (magnesium, phos— phorus, calcium, chloride, sodium and potassium). Listed below are the procedures used. Blood Plasma Ammonia Nitrogen Whole blood was drawn from the jugular vein into a 7 ml. vacutainer tube* using sodium heparin as the anticoagulant. The sample was immedi— ately placed in an ice bath, taken to the laboratory and centrifuged at 10 C. for 10 minutes at 2000 rpm in a model PR2 International Portable Refrigerated Centrifuge.** One milliliter of plasma was pipetted from the sample and placed in a 15 ml. centrifuge tube. The plasma sample, a known nitrogen standard, and a reagent blank were analyzed for plasma ammonia nitrogen by using the Hyland Blood ammonia kit+ as described by + Miller and Rice (1963). In step 7, there was a modificationf of the Hyland test performance in that 2 ml. of 0.02 N NaOH was used. Optical *Becton-Dickinson and Company, Columbia, Nebraska, and Ruther— ford, N.J. **International Equipment Company, Needham Heights, N.Y. +Hyland Division, Travenol Labs, Los Angeles, California. iSuggested by Dr. Robert Foy, Clinical Pathologist, Edward W. Sparrow Hospital, Lansing, Michigan. 13 ”re—'2' l4 densities were determined with the model 6A Coleman Junior Spectropho- tometer* at 630 mu. The formula for plasma ammonia nitrogen determina- tion was: Ammonia nitrogen concen- tration of plasma (pg./100 ml.) Optical Densityiof Sample Optical Density of Standard X 150 = The same spectrophotometer was used in subsequent experiments unless otherwise specified. Prior to the studies on ammonia toxicosis, normal plasma ammonia nitrogen levels were determined by using 18 cattle (Table 1). To establish if a delay in reading intervals would affect the initial plasma ammonia nitrogen levels, plasma samples from 5 Jersey cattle were read at 27 minutes, 3 Jersey cattle at 60 minutes and 18 hours, and 4 Jersey cattle at 10, 30 and 40 minute intervals. To compare the levels of plasma ammonia nitrogen in cattle fed brome grass and orchard grass pasture versus those fed in drylot, 9 Jersey cattle were initially sampled. Five were turned out on pasture and 4 were maintained in the drylot. In 3 days all cattle were rebled and the comparisons recorded. Blood Urea Nitrogen Blood samples for blood urea nitrogen were collected aseptically from the jugular vein with a 5 ml. plastic syringe and transferred to a 5 ml. glass vial containing dipotassium ethylenediaminetetraacetate (EDTA) as the anticoagulant. All samples were refrigerated overnight at 4 C. Determinations were made by using the diacetyl monoxine method as described by Skeggs (1957) and Marsh at al. (1957). This method *Coleman Instruments, Incorporated, Maywood, Illinois. 15 Table 1. Determination of average blood plasma ammonia nitrogen level in cattle Plasma Ammonia Animal Age Nitrogen No. Breed (yrs.) Sex (ug./100 ml.) Experimental cattle l Holstein-Friesian 1 Steer 126.00 2 Holstein-Friesian 1 Steer 148.20 Other cattle 3 Jersey 6 Cow 20.10 4 Jersey 6 Cow 43.80 5 Jersey 4 Cow 63.30 6 Jersey 3 Cow 132.60 7 Jersey 3 Cow 33.00 8 Jersey 1 Heifer 52.95 9 Jersey 5 Cow 99.30 10 Jersey 5 Cow 26.10 11 Jersey 5 Cow 57.30 12 Jersey 2 Heifer 53.17 13 Holstein-Friesian 6 Cow 150.15 14 Holstein-Friesian 5 Cow 207.82 15 Holstein-Friesian 8 Cow 199.69 16 Holstein-Friesian 7 Cow 222.68 17 Holstein-Friesian 3 Cow 160.83 18 Hereford 1 Bull 149.08 Average 108.09 Jersey average 58.16 Holstein—Friesian average 170.50 l6 utilizes the auto analyzer. Samples were read at 480 mu in the spec— trOphotometer and results expressed in mg./100 ml. Blood pH Blood samples for pH determinations were collected from the coccy— geal vein into a 1 ml. plastic syringe containing sodium heparin as the anticoagulant. Immediately, the samples were placed in an ice bath and values determined by using Micro Electrode Unit E5021* as described by Andersen at al. (1968). Since blood pH was to be an important phase of studies on ammonia toxicosis, 10 Jersey cattle were used to determine normal values and to find out if these values would be affected by a change from drylot to brome grass and orchard grass pasture. These cattle were bled, exposed to pasture, and rebled as previously described in the plasma ammonia nitrogen determinations. To compare the effects of delayed versus immediate determination on blood pH value, 2 Guernsey and 4 Holstein-Friesian cattle were bled and sampled immediately. The samples were then maintained at 25 C. and read at 10, 20 and 60 minute intervals. Serum Electrolytes Blood was collected from the jugular vein into 7 ml. vacutainer vials for serum electrolyte determinations (magnesium, phosphorus, cal- cium, chloride, sodium and potassium). The serum was transferred into a 3 ml. glass vial and refrigerated at -4 C. overnight. The serum sample was then thawed at 25 C. and analyzed in the laboratory. *Radometer, COpenhagen, Denmark. 17 Magnesium. Serum.magnesium was determined by the microtechnique of Bohuon (1962). Harleco #25831* was used as the standard. The samples were read in a spectroPhotometer at 510 mu and the results recorded in mEq./L. Phosphorus. Blood serum samples were analyzed by Hycel procedures** with potassium phosphate as the standard. The unknown and standard were spectrOphotometrically read at 650 mu. To form a standard curve, the Optical densities of the standard were plotted against concentra- tion on regular graph paper. These results were recorded in mg./100 ml. Calcium. Serum calcium was determined by the titration method of Backra et a2. (1958) with the titrations made from a known calcium standard. The following formula was used to obtain values which were then reported in mg./100 ml. 1 E TA 1 k X Concentration Standard m . D (t trate un nown) ml. EDTA (standard) = Concentration of calcium in sample Chloride. Serum chloride was analyzed by the method of Schales and Schales (1941). Sodium chloride was used as the standard. The follow— ing formula was used and results reported in mEq./L. Chloride Cone. of Unknown _ Cone. of Standard Concentration of No. ml. Hg(NO3)2 used to - No. of ml. Hg(N03) used a chloride in titrate unknown to titrate standar sample *Hartman-Leddon Company, Inc., Philadelphia, Pennsylvania. **Hycel, Incorporated, P. 0. Box 36329, Houston, Texas. 18 Sodium and Potassium. Sodium and potassium were determined in blood serum by using the Model 21 Coleman Flame Photometer* (Coleman Model 21 Flame Photometer D-248A 1967 Operation Manual). Sterox reagent** was used for both determinations with known sodium and potassium solu- tions as standards. Results were automatically determined on a spectro- photometer and expressed in mEq./L. To determine if brome grass and orchard grass pasture would affect the serum electrolyte values of drylot cattle, 10 Jersey cattle were initially bled. Five of the cattle were turned out on pasture and 5 cattle were maintained in the drylot. After 3 days all cattle were rebled and pasture versus drylot values were compared. Experimental Ammonia Toxicosis Experimental Animals. Two Holstein-Friesian steers, 14 months of age, were used in this research. Both animals were housed in a single 10 x 10 pen. Alfalfa hay was fed ad Zibitum. Preparation of Experimental Ammonium Compounds. The following freshly prepared solutions were used in individual experiments: ammonium car— bonate (1.6%, 4%, 6%, 7%, 8%), ammonium chloride (1%) and ammonium oxalate 63%). These were prepared by mixing the ammonium compound** with 500 ml. of distilled water (Table 2). These compounds were in— jected intravenously to determine their effects on blood pH, serum electrolytes (chloride, sodium, potassium), blood urea nitrogen and plasma ammonia nitrogen levels. *Coleman Instruments, Incorporated, Maywood, Illinois. **J. T. Baker Chemical Company, Phillipsburg, N.J. 19 Table 2. Identification and weight of experimental Holstein-Friesian steers, duration of treatments, ammonium treatments, dates of injections Duration of, * Date of Animal Weight Injection Ammonium Compound Injection No. (kg.) (min.) (%) (1968) l 325 55 1.6 ammonium carbonate 8-1 1 338 42 4.0 ammonium carbonate 8-19 2 346 55 6.0 ammonium carbonate 8-20 2 328 58 7.0 ammonium carbonate 8-21 1 339 60 8.0 ammonium carbonate 8—22 2 338 18 8.0 ammonium chloride 9-2 1 347 17 7.0 ammonium oxalate 9-3** *Volume was 500 ml. for each injection. **Died. Table 3. Weight of Holstein-Friesian steer No. 2, duration of treat- ment, urea levels, method of treatment, dates of treatment Weight Duration of Treatment Urea Levels Method of Date of (kg.) (min.) (Gm.) Administration Treatment (1968) 355 85 158 Mixed in feed 10-11 379 120* 272 Mixed in feed 10-25 and pumped in rumen *Died. 20 Ammonium Compound Injection Technique. A 14 Ga. 2 inch needle was inserted into the jugular vein and the vein cannulated with polyethylene tubing No. 90.* A 20 Ga. 1 inch needle was inserted into the tubing and connected to a gravity flow intravenous outfit which had previously been connected to the plastic bottle containing the ammonium mixture. During the ammonium chloride and ammonium oxalate injections, an 18 Ga. 1-1/2 inch needle was used in place of the 20 Ga. 1 inch needle. There were a total of 7 experimental injections in which steer No. l was injected 4 times and steer No. 2 injected 3 times. Steer No. 1 died during the infusion of 7% ammonium oxalate. Blood pH. To determine the effect of ammonium compounds on blood pH, steer No. 1 was injected intravenously with 4% ammonium carbonate and 7% ammonium oxalate and steer No. 2 injected intravenously with 6% ammonium carbonate. Serum Electrolytes. Injections of 4% ammonium carbonate and 8% ammonium carbonate were administered intravenously to steer No. 1 to determine their effect on initial serum electrolyte values (chloride, sodium, potassium). Blood Urea Nitrogen. To assess the influence of ammonium carbonate on blood urea nitrogen, steer No. 1 was injected intravenously with the following solutions: 1.6% ammonium carbonate, 4% ammonium carbonate and 8% ammonium carbonate. Plasma Ammonia Nitrogen. Plasma ammonia nitrogen levels were an impor- tant phase in the experimentally produced ammonia toxicosis. These *Clay Allen, Incorporated, New York, N.Y. 21 levels could serve as a diagnostic aid in suspected toxicosis from ammonia producing compounds. To establish the influence on plasma ammonia nitrogen levels, steer No. l was injected intravenously with 1.6% ammonium carbonate, 4% ammonium carbonate, 8% ammonium carbonate and 7% ammonium oxalate. Steer No. 2 was similarly injected with 6% ammonium carbonate, 7% ammonium carbonate and 8% ammonium chloride. Urea Experiments To terminate the investigation and compare the effects of ammonium compounds and urea, steer No. 2 was treated orally with 2 levels of feed-grade urea* (Table 3). Collection of blood and laboratory deter- minations were the same as previously described. The effects of urea on blood pH, blood urea nitrogen and plasma ammonia nitrogen levels were studied and compared with levels obtained following earlier intra- venous injections of ammonium compounds. For a period of 12 days prior to the first treatment, steer No. 2 was fed a diet of 1150 Gm. of corn, 1150 Gm. of oats, 916 Gm. of molasses and alfalfa hay was fed ad Zibitum. Twenty-four hours before treatment, the diet was halved. On the morning of the treatment, 158 Gm. of urea was added to the whole diet and fed to the steer. All feed was consumed. The same diet was fed for a similar period prior to the second treatment. Eighteen hours before this treatment all water was withheld. On the morning of the treatment, 286 Gm. of 44% soybean oil meal and 250 Gm. of urea were added to the whole diet and fed to the steer. After a lS-minute interval, the steer refused to eat. At that time, 166 Gm. of urea was mixed with 2 liters of tap water and administered by stomach pump through a stomach tube *Eastman Organical Chemicals, Rochester, N.Y. 22 into the rumen. A11 feed not eaten by the steer was weighed to determine the total urea intake. Before death, a total of 272 Gm. of urea had been administered. Necropsy Following the death of both steers, a necropsy was performed. Organs examined were kidneys, cerebellum, spinal cord, heart, abomasum and liver. Tissues were fixed in 10% buffered formalin, processed routinely and paraffin sections were cut at 6 mu and stained by the hematoxylin-eosin method (Armed Forces Institute of Pathology Manual of'HistoZogic and Special Staining Téchnics, 1957). RESULTS When whole heparinized blood samples were collected from Jersey cattle and maintained at 25 C. and analyzed at various time intervals, there was a marked decrease in blood plasma ammonia nitrogen levels (Tables 4, 5 and 6). The data indicated that testing or analyzing for blood plasma ammonia nitrogen levels should not be delayed longer than 30 minutes after the initial collection, to ensure accuracy. Pasturing Jersey cattle on brome grass and orchard grass for a period of 3 days caused a marked increase in blood plasma ammonia nitrogen levels (Table 7). The results indicated that diet can affect blood plasma ammonia nitrogen levels. Blood pH values were recorded in 10 Jersey cattle (Table 8). The results indicated that the normal blood pH values of these cattle was 7.32. The effects of access to pasture versus maintenance in a drylot are illustrated (Table 8). The results indicated there was only a minimal effect on blood pH. Delay- ing the readings for blood pH until 10, 20 or 23 minutes after the initial reading caused only slight alteration from initial blood pH values in Guernsey and Holstein—Friesian cattle (Table 9). The effects of access to brome grass and orchard grass pasture on the blood serum magnesium, phosphorus, calcium, chloride, sodium and potassium levels of Jersey cattle are shown (Table 10). The results indicated that the brome grass and orchard grass pasture caused only minimal changes from the initial values of these blood serum electrolytes. 23 24 Table 4. Effect of delayed reading time on blood plasma ammonia nitro- gen levels in Jersey cattle Animal Initial Sample 27 Minute Time* Interval No. (ug./100 ml.) (ug./100 ml.) 4 43.80 34.95 7 33.00 30.30 8 26.70 20.50 10 27.90 18.90 12 44.55 31.35 Average 35.19 27.20 *Paraffin placed on sample after initial reading. Table 5. Effect of delayed reading time on blood plasma ammonia nitro- gen levels Time Interval 60 18x7 Animal Initial Sample (min.) (hr.) No. (ug./100 m1.) (ug./100 ml.) 5 63.30 63.30 49.20 9 99.30 91.20 73.80 11 57.30 57.30 47.85 Average 73.30 70.26 56.95 *Paraffin placed on sample after reading and at 7 C. sample refrigerated 25 Table 6. Effect of delayed reading time on blood plasma ammonia nitro— gen levels )7 Minute Time Interval Animal Initial Sample .1—0"F 30* 40** No. (ug./100 ml.) (ug./100 ml.) 6 132.60 128.85 124.20 120.90 8 79.20 73.95 67.95 63.15 10 24.30 18.75 18.75 15.00 12 61.80 58.50 53.85 51.45 Average 74.45 70.01 66.18 62.62 *Paraffin placed on sample after initial reading. **Paraffin removed from sample after second reading. 26 Table 7. Effect of brome grass and orchard grass pasture on blood plasma ammonia nitrogen levels in Jersey cattle Animal Initial Sample Drylot Sample Pasture Sample No. (ug./100 ml.) (ug./100 m1.) (pg./100 ml.) 3 28.10 79.35 --- 4 43.80 74.40 _-_ 5 63.30 82.50 -—- 6 132.60 64.05 --- 8 52.95 --- 219.75 9 99.30 --- 166.65 10 26.10 --- 234.60 11 57.30 --- 160.20 12 53.17 --- 125.55 Average 61.84 75.07 181.35 27 Table 8. Effect of brome grass and orchard grass pasture on blood pH of Jersey cattle Animal No. Initial Sample Drylot Sample Pasture Sample 3 7.26 --—* --- 4 7.36 7.23 ___ 5 7.30 7.23 --— 6 7.31 7.30 —-- 7 7.31 7.38 --- 8 7.32 —-— 7.40 9 7.38 -—— 7.40 10 7.32 —-- 7.35 11 7.38 ——- 7.35 12 7.30 —-- 7.14 Average 7.32 7.28 7.29 *Escaped from drylot. 28 Table 9. Effect of delayed reading on blood pH in Guernsey and Holstein-Friesian cattle if Animal Initial Time Interval in Minutes No. Breed Sample 10 20 60 19 Guernsey 7.46 7.45 7.47 7.47 20 Guernsey 7.36 7.34 7.33 7.35 21 Holstein-Friesian 7.36 7.34 7.35 7.37 22 Holstein-Friesian 7.36 7.36 7.33 7.35 23 Holstein-Friesian 7.44 7.38 7.38 7.39 24 Holstein-Friesian 7.45 7.34 7.42 7.44 Average 7.40 7.38 7.38 7.39 29 mauououu wo>amoaa unflaaauoe H.q mma moa 0.0a N.n N.N owmuo>< H.¢ «ma NON m.oa m.¢ N.N N NH N.m OMH moa m.m H.m 0.N 0 NH H.¢ Nma moa ¢.m 0.0 m.N 0 OH m.q Hma NoH m.HH m.m o.m m m m.q mma moa m.m 0.0 m.N H w whoa mIIoHQEMm ounummm m.q NMH moa N.OH 0.0 m.N owmuo>< N.q 00H moa a.m 0.0 N.m m N N.¢ oqa moa <.m m.N m.N m 0 «.0 NmH moa N.oa o.N m.N a m H.q 00H ma m.oa 0.0 o.N 0 a II: III III III III III N am when mnnoaafimm chow m.¢ wma Noa m.a N.m m.N owmuu>¢ 0.0 aca «Ha m.m m.q N.N N NH N.¢ mma 00H m.oa H.n m.N 0 NH 0.0 Nma moa H.OH 0.0 n.N m 0H N.q Nma 00H o.oa o.0 H.N m m 0.0 0ma 00H 0.0H 0.N m.N H m 0.0 and 00H 0.0H N.N m.N m N N.m Had HHH 0.m «a0.m m.N m 0 0.0 Nqa oaa m.oa m.N m.N a m 0.0 and 00H 0.m «.5 N.N 0 a m.¢ Nma «Ha 0.0 eo.N m.N N m oaaamm HmHuHeH . Aa\.amav Aa\.sma0 Aq\.umav A.Ha ooH\.mav . A.Ha ooa\.wav Aa\.emav A.usv .oz abammMuom azapom ovfiuoano aufioamo msuonamonm anamoawmz ow< Hmaao¢ ”up!!! FIL LHIA Ill! oauuwo hookah mo mau>oa anaemMuoa new abfivom .owauoaso .abfioamo .mouosn Imoza .asamoawma.aauom vooan do «gunman mmmuw vumnuuo pom mmmuw oaoun Ou ounmomxo mo uoummm .OH manna 30 The results of the intravenous infusion of steer No. 1 with 4% ammonium carbonate and 7% ammonium oxalate and steer No. 2 with 6% ammonium carbonate and their effects on blood pH are illustrated (Table 11). The data indicated that these ammonium compounds caused only a slight elevation of blood pH values. The effects of the intravenous injections of 4% ammonium carbonate and 8% ammonium carbonate on the blood serum chloride, sodium and potassium levels of steer No. 1 are recorded (Table 12). Ammonium carbonate had only a minimal effect on blood serum chloride, sodium and potassium. The results of the intravenous infusions of 1.6% ammonium carbonate, 4% ammonium carbonate and 8% ammonium carbonate on the blood urea nitro— gen levels of steer No. l are illustrated (Table 13). Treatment with ammonium carbonate resulted in blood urea nitrogen levels which were essentially the same as the initial levels. The effects on blood plasma ammonia nitrogen levels following the intravenous infusions of steer No. l with 1.6% ammonium carbonate, 4% ammonium carbonate, 8% ammonium carbonate and 7% ammonium oxalate and steer No. 2 with 6% ammonium carbonate, 7% ammonium carbonate and 8% ammonium chloride are recorded (Table 14). The results indicated these ammonium compounds caused a marked increase in plasma ammonia nitrogen levels which were directly preportional to the percentage of ammonium compound injected. Steer No. 1 died following the intravenous infusion of 7% ammonium oxalate. Blood plasma ammonia nitrogen levels were a reliable indication of the severity of ammonia toxicosis. The effects of the administration of 158 Gm. and 272 Gm. of urea on blood pH values of steer No. 2 are illustrated (Table 15). Both levels of urea caused a slight elevation of the initial blood pH values. 31. Table 11. Effect of the intravenous injection of 500 milliliters of 4% ammonium carbonate, 6% ammonium carbonate, and 7% ammonium oxalate on the blood pH of Holstein-Friesian steers Ammonium Duration of Shortly Immediate- Animal Weight Compound Infusion Before 1y After No. (kg.) (%) (min.) Infusion Infusion l 338 4% ammonium carbonate 42 7.34 7.40 2 346 6% ammonium carbonate 55 7.30 7.35 1 347 7% ammonium oxalate 17 7 .30 7 . 32* Average 7.31 7.35 *Died. 32 m .m m: No .70 a: No.” 0 00 mmm NNIw 0.0 N: co.” m .1» mma mm a Na 0mm 310 E . was a: ems 8. 735 Two: 38: anammMuom anfivom ovfiuoago esfimmmuom asfiwom ovfiuoaso oumconumu scamomoH usmfioz uooaumouH «Ammdm nowuuwfldfiumom camamm HmHuHaH anacoaa< mo oofiumuon mo uuma H .02 Hooum cmfimoauhlcfioumaom a“ mHo>oH asammmuom was aofivom .ovauoaso abuow vooan any do oumaonumo sowcoaam N0 0am «unconumo aufiaoaam N0 mo mucuaafiaaaa com mo aowuoomaa mooou>muuafi «no mo uuommm .NH manna 33 Table 13. Effect of intravenous injection of 500 milliliters of 1.6% ammonium carbonate, 4% ammonium carbonate, and 8% ammonium carbonate on the blood urea nitrogen levels in Holstein- Friesian steer No. l Date of Injection Ammonium Initial Postinjection Injection Time weight Carbonate Sample Sample (1968) (min.) (kg.) (%) (mg./100 ml.) (mg./100 ml.) 8-1 55 325 1.6 16 16 8-19 42 338 4 4 6 8-22 60 339 8 19.5 20 34 Table 14. Effect of intravenous injection of 500 milliliters of 1.6% ammonium carbonate, 4% ammonium carbonate, 6% ammonium carbonate, 7% ammonium carbonate, 8% ammonium carbonate, 8% ammonium chloride and 7% ammonium oxalate on blood plasma ammonia nitrogen levels in Holstein-Friesian steers Infusion Infusion Ammonium Initial Postinfusion Animal Time Date Compound Sample Sample No. (min.) (1968) (%) (ug./100 ml.) 1 55 8-1 1.6 ammonium carbonate 148.20 278.80 1 42 8-19 4.0 ammonium carbonate 141.90 445.20 2 55 8-20 6.0 ammonium carbonate 126.00 732.00 2 58 8-21 7.0 ammonium carbonate 208.50 1205.70 1 60 8—22 8.0 ammonium carbonate 190.35 1319.40 2 18 9-2 8.0 ammonium chloride --- 1484.70 1 17 9-3 7.0 ammonium oxalate ——- 1507.50* *Died vufinaa vouhamumma seam III amN Ha NNN mum ONH III 0H III a mma 00m mm 03 TH omfmav 8 use 8265 750 795 73.5 oaaamm coauovfiaaumom «Haamm Ho>oq unmaoz uaoaumoua mouscaz cw Hm>uoucH cage HmHuHoH «on: we coaumuao N .02 nooum anamoaumIoHoumHom mo Ho>oH cowouufia mop: wooan can :0 mos: mo uoommm .0H «dome 5 3 peace «Nm.0 III om.n mN.N CNN NNN mum nNIoa III om.N III 0H.N mm 00H nmm HHIoa 8a 3 I8 oaaaom Hand: dons 3.8 Add 333 mousafiz cw Hm>uouomemaagamm uaoaumoua Ho>og unwfio3 coauwuumacwav< mo coaumusn mono mo coma N .02 Hooum amamoaumlafioumaom no me mooan «nu do «on: mo cowumuumfiawamm 03» mo uoommm .mH «HAMH 36 Death resulted after the administration of 272 Gm. of urea. Blood pH decreased just prior to death of steer No. 2. Blood urea nitrogen levels of steer No. 2 were changed at 60, 85 and 120 minutes as a result of feeding 158 Gm. and 272 Gm. of urea (Table 16). The data indicated that feeding of these levels of urea will cause a marked increase in blood urea nitrogen. The effects on blood plasma ammonia nitrogen levels due to the oral administration of 158 Gm. and 272 Gm. of urea to steer No. 2 are recorded (Table 17). The results indicated that high levels of urea caused a marked increase in initial blood plasma ammonia nitrogen levels. Clinically ammonium compounds and urea produced essentially the same toxic signs. These signs were rumen atony, muscle tremors, fre- quent urination, shallow respiration, ataxia, tachycardia, tetany, clonic convulsions, paralysis and death. The signs in Holstein- Friesian steer No. iffollowing the administration of 272 Gm. of urea are shown (Figures 1, 2, 3 and 4). Characteristic gross or microsc0pic lesions were not found in steer No. 1 that died as a result of 7% ammonium oxalate. Marginal perivascular and perineuronal edema in all sections of the central nervous system and edema of the lamina propria of the abomasal wall was present on necropsy of steer No. 2 that died from a high level of urea o 37 moans amaou o£u coca mousse was «00.0N0m III oa.0mHN mN.NmH NNN poem ca voxfiz ONH mum nNIoa III 0H.o00 III No.00a 00H menu a“ cox“: mm mnm HHIOH TE 8: .me nae 81.20 :50 aoaoonaoaoaaoa 3:3 Two: 333 ONH 00 O0 H0>og Ho>uq mo vogue: uooaumouH unwfioa ucoaumoua mouaaflz ea Ho>uouaH mafia HmfiufiaH may: mo aofiumuaa mo oumn N .02 “ovum amHmoHHMIafioumHom ca Ho>oa cowouufic macoaam mammam vooan can so mun: mo uoommm .NH MHQMH 38 1 Figure l. Holstein-Friesian steer No. if, 15 minutes after administration of 272 Gm. of urea. 39 Figure 2. Initial urea toxicosis in Holstein-Friesian steer No. , 30 minutes after administration of 272 Gm. of urea. 40 Figure 3. Urea toxicosis. Increasing toxic signs 45 minutes after administration of 272 Gm. of urea to Holstein-Friesian steer No. 2. 41 Figure 4. Urea toxicosis. Paralysis prior to death of Holstein-Friesian steer No..g following administration of 272 Gm. of urea. Sixty minutes after administration. DISCUSSION This research was conducted to clinically evaluate ammonia toxi— cosis in Holstein-Friesian steers. The data indicated that ammonium compounds injected intravenously or urea administered orally could result in toxicosis and death in Holstein-Friesian steers. The research further indicated that the severity of the toxicosis was dependent on the percentage of ammonium compound injected and the level of urea administered. The physiological effects were compared. Ammonium compounds and urea produced essentially the same toxic signs. The signs observed were rumen atony, muscle tremors, polyuria, shallow respiration, ataxia, tachycardia, tetany, clonic convulsions, paralysis and death. These signs were similar to those reported in cattle (Whitehair, 1961), in pigs (Bicknell, 1966) and in dogs and sheep (Wilson et al., 1968). Additional toxic signs observed in the urea experiments were flexion of the pasterns, extension of the fore and hind limbs and apparent blindness. As the toxic signs were essentially the same following the intra- venous injections of ammonium salts of carbonate, chloride and oxalate and administration of urea into the rumen this research suggests that the ammonium ion was the cause of toxicosis. This had been suggested in ammonia toxicosis studies in sheep (Lewis, 1960; Wilson et al., 1968) and in pigs (Bicknell, 1966) and in dogs (Wilson at aZ., 1968). These results did not confirm the conclusions of Kaishio et a1. (1951) and Hale and King (1955) in sheep that ammonium carbamate was the toxic 42 43 intermediate. Lewis (1960) questioned the stability of ammonium car- bamate and Wilson at al. (1968) were of the Opinion that had these authors used ammonium carbonate in place of ammonium carbamate the same toxic effects would have resulted. Since this research involved the intravenous injections of 3 ammonium compounds, the carbamate ion would not be formed since the rumen was bypassed, therefore lending support to the theory that the ammonium ion is the cause of toxicosis. Salivation was observed in lambs and steers following administra- tion of 20 Gm. of urea per 40 kg. body weight (Dinning at al., 1948) but was not observed in my research. This could be due to a difference in diet. Rumen atony was the first clinical sign of ammonia toxicosis. The degree of rumen atony was dependent on the percentage of ammonium com- pound injected. At the highest percentage, atony was complete in 5-10 minutes after the injection was started and remained during the injec- tion. Normal rumen contractions returned within 17 to 25 minutes after the injections ceased. This would indicate that the ammonium ion has only a temporary effect on rumen contractions under such experimental procedures. Further research should be conducted to determine the cause of this atony. Urea administered to Holstein-Friesian steer No. 2 resulted in rumen. atony within 22 minutes following the administration, which would agree with similar work in sheep (Annicolas at aZ., 1956). In steer No. 2, which died from a high level of urea, the rumen pH increased above 8. This increased pH was in agreement with urea experiments in sheep (Coomber and Tribe, 1958). Hogan (1961), in similar experiments, indi- cated that as the pH increases in the rumen there is an increased transfer of ammonia across the rumen epithelium. To decrease pH may be 44 the rationale for using acetic acid to treat urea toxicosis. Bloating as reported in consumption of high levels of urea in sheep (Gallup at aZ., 1953) and in dairy cattle (Bullington et al., 1955) was not observed in this research. This indicated that other factors than rumen atony were necessary to cause bloating or there was a difference in diets fed. Muscle tremors were an early manifestation of ammonia toxicosis and initially involved the triceps muscles. The severity of the tremors and the involvement of the skeletal musculature were directly propor- tional to the percentage of ammonium compound intravenously injected or the level of urea administered orally. Following urination there was a temporary decrease in muscle tremors. As tremors were the first external sign of ammonia toxicosis, they would be a good clinical diag— nostic aid as to the severity of the ammonia toxicosis. Shallow respirations were observed as a clinical sign of ammonia toxicosis. In both steers that died, the respiratory centers were paralyzed prior to death. The increased respiration was possibly an attempt on the steer's metabolic system to eliminate excess hydrogen ions. The increase in heart rate was directly preportional to the per- centage of ammonium compound injected or the level of urea administered. The marked tachycardia and ventricular fibrillation prior to death indicated that the ammonium ion had a direct cardiotoxic effect. This was suggested by Lewis (1960) in urea experiments in sheep. Similar conclusions were indicated by Wilson et al. (1968) following the intra- venous injection of ammonium carbamate in a decerebrated and bilaterally vagotomized dog. 45 Polyuria occurred in the ammonia toxicosis experiments, and the frequency was directly proportional to the percentage of ammonium com- pounds injected or level of urea administered. The temporary decrease in muscle tremors following urination has not been reported in the literature and was an outstanding finding in this research. Certain ammonium compounds have a diuretic action due to their osmotic effect on the body electrolytes. Urea, which is poorly reabsorbed from the tubules of the kidneys, attracts water from the body and thus has a diuretic action. This temporary decrease in muscle tremors following urination would provide a good problem for additional research in ammonia toxicosis. The blood plasma ammonia nitrogen levels were a good indication of the degree of ammonia toxicosis. These results were in agreement with ammonia toxicosis studies in sheep (Lewis, 1960). Death occurred when the peripheral blood levels were between 1.4 and 3.2 mg./100 ml. The levels were essentially the same as reported in sheep (Lewis, 1960) and in protein experiments in dairy cattle (Holzachuh and Wetterau, 1962). If procedures are properly carried out, blood plasma ammonia nitrogen levels would serve as a diagnostic aid in suspected cases of ammonia toxicosis. The results indicated that to ensure accuracy, the plasma sample must be immediately analyzed following completion of the test procedures. Ammonium compounds had little effect on blood urea nitrogen levels, whereas urea caused a marked increase. As these levels were compatible with life, the results indicated that blood urea nitrogen levels were not a good indication of the severity of ammonia toxicosis. Davis and Roberts (1954), in their urea experiments in cattle, indicated blood urea nitrogen levels of 22 mg./100 m1. caused no toxic effects, while 46 c death and toxitosis occurred when the levels reached 42 mg./100 ml. The increase in blood urea nitrogen following the administration of urea would indicate that all urea was not hydrolyzed and excess urea dif- fused across the rumen wall. This is in agreement with Dinning at al. (1948) in urea experiments in sheep and cattle. The absence of characteristic gross and microsc0pic lesions in steer No. 1 that died from ammonium oxalate injection indicated that this compound produced an acute toxicosis. Frequently in an acute toxi- l .. cosis lesions are absent. The findings in this experiment were in agreement with ammonia toxicosis in pigs (Bicknell, 1966). In steer No. , w 2 that died from the administration of urea, the diffuse hemorrhages on a the mucosal surface of the abomasum were due to the irritating preper- ties of excess urea. Essentially the same findings were reported in the urea pathology in ruminants (Fujimoto and Tajima, 1953). The ecchy- motic hemorrhages noted on the endocardium and myocardium were in agreement with similar lesions reported by Clark at al. (1951) in urea toxicosis studies in sheep. Histologically both steers had a toxic nephrosis which indicated that the excessive urea eliminated by the kidneys was causing degenera— tive changes. In steer No. 2 the perivascular and perineuronal edema in all sections of the central nervous system could be possibly corre- lated with the convulsions noted in this animal prior to death. In this research, the steers either died within 2 hours or recovered. During the course of the experimental procedures little effect was noted on their appetite or their weight gain. These results indicated that ammonium compounds or urea did not have a permanent effect on surviving animals. 47 The minimal effect on blood pH from administration of urea or injection of ammonium compounds indicated that the acid base balance of the blood was not a factor in ammonia toxicosis. Similar findings were noted on blood serum chloride, sodium and potassium following the injection of an ammonium compound. Additional Procedures These procedures were not a part of my research; however, during I the experimental procedures additional procedures were carried out. The results indicated in reading blood pH immediate determinations are not necessary to ensure accuracy. 5 In the brome grass and orchard grass pasture determinations, the results indicated that these grasses did not have an effect on blood pH. Initial serum electrolyte levels were not affected by the intra- venous injection of ammonium carbonate, indicating that serum electro- lytes are not altered in ammonia toxicosis. Serum electrolytes were not affected by exposure to brome grass and orchard grass pasture and therefore these grasses are probably not a factor in regard to calcium, phosphorus and magnesium in muscle tetany in conditions associated with lush spring pasture. Jersey cattle exposed to brome grass and orchard grass pasture resulted in a marked increase in blood plasma ammonia nitrogen levels over those maintained in drylot. These levels were not much higher than levels determined in other cattle. These results indicated that the pasture grass was rich in protein content. SUMMARY Toxicosis and death were produced in Holstein-Friesian steers by the intravenous injections of ammonium compounds and the administration of urea into the rumen. The severity of the toxicosis was directly pr0portiona1 to the level of compound given. When the peripheral blood plasma ammonia nitrogen levels reached 0.7 mg./100 ml., toxicosis was noted and death occurred when levels were between 1.5 and 3.2 mg./100 ml. Signs of toxicosis were essentially the same for both the ammonium compounds and the urea. Toxic signs observed were ataxia, rumen atony, muscle tremors, polyuria, shallow respiration, tachycardia, paralysis of the Holstein-Friesian steers, convulsions and death. Additional toxic signs noted with high urea levels were flexion of the pasterns, extension of the legs and apparent blindness. There was a temporary decrease in muscle tremors following urination. The Holstein-Friesian steers died following ventricular fibrilla- tion which indicated that the ammonium ion had a direct toxic effect on the heart. Since similar toxic signs were caused by the administra- tion of ammonium compounds and urea, it was concluded that the ammonium ion was the cause of death of the Holstein-Friesian steers and the cause of the syndrome known as urea toxicosis. Twenty to thirty Grams of urea per 40 kg. of body weight will pro- duce toxicosis and death in Holstein-Friesian steers. Following ingestion of toxic levels of urea, animals will usually become sick and die within 48 jififi’ 49 2 hours or recover. Intravenous injections of 0.8 mM/kg. of ammonium carbonate produced toxicosis; however, the animals survived, while 0.6 mM/kg. of ammonium oxalate was fatal. On necrOpsy, no characteristic gross or microsc0pic lesions were observed in the steer that died from the intravenous injection of ammonium oxalate. Perivascular and perineuronal edema of the central nervous system, ecchymotic hemorrhages of the mucosa of the abomasum, and vacuolization of the chief cells of the cortex of the kidneys were ~‘ present on necropsy of the steer that died from a high urea level. ‘- Blood plasma ammonia nitrogen levels in Holstein-Friesian steers increased in direct pr0portion to the amount of ammonium compound injected or the amount of urea administered into the rumen. These levels could be used as a diagnostic aid in cases of suspected ammonia toxicosis from the overconsumption of urea. By maintaining plasma samples at 25 C. and analyzing at delayed time intervals there was a marked decrease in ammonia nitrogen levels, indicating the importance of reading samples immediately after completing the ammonia nitrogen test procedures. Initial blood urea nitrogen levels of Holstein-Friesian steers were increased after the administration of ammonium compounds and urea. However, these levels were compatible with life and therefore were not a reliable indication of the severity of ammonia toxicosis. Intravenous injections of ammonium compounds in Holstein-Friesian steers resulted in a blood pH increase. Similar effects were noted with urea administration initially; however, the blood pH decreased prior to death. Maintaining blood samples of Jersey cattle at 25 C. and analyzing at delayed time intervals caused little alteration of blood pH values. Pasturing of Jersey cattle on brome grass and 50 orchard grass pasture caused only a slight increase above initial values. Injections of ammonium compounds had little effect on serum chlor- ide, sodium and potassium levels of Holstein-Friesian steers. Jersey cattle were pastured on brome grass and orchard grass pasture for a period of 3 days with minimal increase in serum magnesium, phosphorus, calcium, chloride, sodium and potassium. —— WEEK REFERENCES Andersen, 0. S., Engel, K., Jorgenson, J., and Astrup, P. 1968. A micro method for determination of pH, carbon dioxide tension, base excess and standard bicarbonate in capillary blood. Accepted for publication. Scand. J. Clin. Lab. Invest. Annicolas, D., Le Bars, H., Nugues, J., and Simonnet, H. 1956. Studies on the motility of the rumen in.small ruminants. 6. Effect of ingestion of urea. 7. Effect of diet on the inhibition of rumen motility induced in the sheep by ingestion of urea. Bull. Acad. Vet. France, 29: 257—261, 263-265. Armed Forces Institute of Pathology. 1957. Manual of'Histologic and Special Staining Technics. Washington, D.C. Backra, B. N., Dauer, A., and Sobel, A. E. 1958. The complexometric titration of micro and ultramicro quantities of calcium in blood stream, urine and inorganic salt solutions. Clin. Chem., 4: 107-109. Bartley, E. E., Deyoe, C. W., and Pfost, H. B. 1968. An improved urea product for ruminants. Anim. Nutr. Health, May: 10-13. Bessman, S. P., and Bessman, A. N. 1955. Cerebral and peripheral up- take of ammonia in liver disease with a hypothesis for the mechanism of hepatic coma. J. Clin. Invest., 34: 622-628. Bicknell, E. J. 1966. Experimental ammonia toxicosis in the pig. Diss. Abstr., 27, Part 1: 863B. Blaser, V. E. 1962. The treatment of liver coma in cows. Berl. Munch. Tierartztl. Wsch., 75: 381-382. Bloomfield, R. A., Garner, G. B., and Muhrer, M. E. 1960. Kinetics of urea metabolism in sheep. J. Animal Sci., 19: 1248. Bohuon, C. 1962. .Microdbsage du magnesium dans divers milieux Biologiques. Clin. Chim. Acta, 7: 811-817. Bullington, T. H., Byrd, C. E., and Harris, T. W. 1955. Urea poison- ing in the bovine. No. Amer. Vet., 36: 107-109. Chalupa, W. 1968. Problems in feeding urea to ruminants. J. Anim. Sci., 27: 207-219. 51 52 Clark, R., Oyaert, W. A., and Quin, J. I. 1951. Studies on the ali- mentary tract of the Merino sheep in South Africa. XXI. The toxicity of urea to sheep under different conditions. 0nderste- poort J. Vet. Res., 25: 73—78. Conn, H. O. 1960. Ammonia tolerance in liver disease. J. Lab. Clin. Med., 55: 855-871. Coomber, J. B., and Tribe, D. E. 1958. Toxicity of urea to sheep. Nature, 182: 116-117. Davis, G. K., and Roberts, H. F. 1954. Levels of blood urea in urea feeding in cattle. Proc. Assoc. Southern Agric. WOrkers, 51: 73-74 0 Dinning, J. S., Briggs, H. M., Gallup, W. D., Orr, H. W., and Butler, R. 1948. Effect of orally administered urea on the ammonia and urea concentration in the blood of cattle and sheep with obser- vations on blood ammonia levels associated with symptoms of alkalosis. Am. J. Physiol., 153: 41-46. Eiseman, B., Fowler, W. G., White, P. J., and Clark, G. M. 1955. The role of ammonia in the production of hepatic coma. Surg. Forum, 6: 369-373. Fujimoto, Y., and Tajima, M. 1953. Pathological studies on urea poison- ing. Jap. J. Vet. Sci., 15: 133-134. Gallup, W. D., Pepe, L. S., and Whitehair, C. K. 1953. Urea in rations for cattle and sheep. Bull. Okla. Agric. Exp. Sta., B409: 7-35. Garner, R. J. 1967. Veterinary Tbxicology. 2nd ed. The Williams and Wilkins Co., Baltimore. Hale, W. H., and King, R. P. 1955. Possible mechanism of urea toxicity in ruminants. Proc. Soc. Exptl. Biol. Med., 89: 112-114. Harris, L. E., and Mitchell, H. H. 1941. The value of urea in the synthesis of protein in the paunch of the ruminant. II. In growth. J. Nutr., 22: 183-196. Hart, E. B., Bohstedt, G., Deabald, H. J., and Wegner, M. I. 1938. The utilization of simple nitrogenous compounds such as urea and ammonium bicarbonate by growing calves. J. Dairy Sci., 22: 785-798. Haupt, T. R. 1958. Utilization of blood urea in ruminants. Diss. Hogan, J. P. 1961. The absorption of ammonia through the rumen of the sheep. Aust. J. Biol. Sci., 14: 448-460. 53 Holzschuh, W., and Wetterau, H. 1962. Untersuchungen uber den abbau von nicht. Protein striksZofTDerbindangen in-Pansen der wuderkauer. Arch. Tierernahr., 3: 161-178. Juhasz, B. 1965. Endogenous nitrogen cycle in ruminants. Acta Veteri- naria Acad. Sci. Hung., 15: 25. Kaishio, Y., Higaki, S., Horii, S., and Awai, Y.. 1951. The transition of administered urea in the body of ruminants. Bull. Nat. Inst. Agric. Sci. (Japan), 2: 131-140. Kirk, E. 1936. Amino acid and ammonia metabolism in liver diseases. Acta Med. Scand. Suppl. 77, 89: 8-147. Koenig, H., and Koenig, R. 1949. Production of acute pulmonary edema by ammonium salts. Proc. Soc. Exptl. Biol. Med., 70: 375-380. Lewis, D. 1960. Ammonia toxicity in the ruminant. J. Agric. Sci., 55: 111-117. Lewis, D., Hill, K. J., and Annison, E. F. 1957. Studies on the portal blood of sheep. I. Absorption of ammonia from the rumen of the sheep. Biochem. J., 66: 587-592. Marsh, W. H., Fingerhut, B., and Kirsch, E. 1957. Determination of. urea nitrogen with the diacetyl method and an automatic dialyzing apparatus. Amer. J. Clin. Path., 28: 681-688. McDermott, W. V., Jr. 1957. Metabolism and toxicity of ammonia. New McDonald, I. W. 1948. The absorption of ammonia from the rumen of the sheep. Biochem. J., 42: 584-587. McGirr, J. L., Papworth, D. 8., and Parr, W. H. 1954. Ammonia sulphate poisoning. Vet. Res., 66: 553-554. McKay, E. M., Jordan, M. D., and McKay, E. L. 1949. Experimental pul- monary edema. II. Pathogenesis of pulmonary edema caused by the ammonium ion. Proc. Soc. Exptl. Biol. Med., 72: 421-424. Miller, G. E., and Rice, J. D., Jr. 1963. Determination of the concen- tration of ammonia nitrogen in plasma by means of a simple ion exchange method. Amer. J. Clin. Path., 39: 97-103. Mills, R. C., Lardinois, C. C., Rupel, I. W., and Hart, E. B. 1944. Utilization of urea and growth of heifer calves with corn molas- ses or cane molasses as the only readily available carbohydrate in the ration. J. Dairy Sci., 27: 571-578. Nencki, M., Pawlow, J. P., and Zaleski, J. 1896. Uber den ammoniagehalt des BZutes und der organs and die Harnstoffbildung bei den Saugetieren. Nauyn-Schmiedeberg's. Arch. Path. Pharmakol., 37: 26. 54 Nicoletti, P. Uren, A. W., and Dale, H. E. 1956. Ammonium chloride as an agent to acidify bovine urine. J. Amer. Vet. Med. Assn., 129: 332-334. Nix, R. R., and Anthony, W. B. 1965. Urea-lethal dose and toxic syndrome for sheep. J. Anim. Sci., 24: 286. Oltjen, R. R., Waller, G. R., Nelson, A. B., and Tillman, A. D. 1963. Ruminant studies with diammonium phosphate and urea. J. Anim. Sci., 22: 36-42. Osebold, B. S. 1947. Urea poisoning in cattle. No. Amer. Vet., 28: 89-91. Perea, P. D., and Nelson, R. S. 1964. Ammonia levels in primary and metastatic cancers of the liver. Clin. Chim.Acta, 9: 13-18. Phear, E. A., Sherlock, S., and Summerskill, W. H. J. 1955. Blood ammonium levels in liver disease and hepatic coma. Lancet, 1: 836-840. Phillips, G. B., Schwartz, R., Gabuzda, G. J., and Davidson, C. S. 1952. Syndrome of impending hepatic coma in patients with cirrhosis of the liver given certain nitrogenous substances. New Eng. J. Med., 247: 239-246. Phillipson, A. T. 1964. The digestion and absorption of nitrogenous compounds in the ruminant. Mammalian protein metabolism. I. Munro, H.N. and Alluson, J. B. Editors, Academic Press, New York. Pierson, R. E., and Aanes, W. A. 1959. Urea poisoning in ruminants. The Allied Veterinarian, 4: 136—139, 156. Pope, L. S., Gallup, W. D., and Whitehair, C. K. 1950. The effect of urea on vitamin A storage in mature ewes. Okla. Agric. Exp. Sta., MP-17: 5-7. Post, R. L., and Jolly, P. C. 1957. Linkage of sodium, potassium and ammonium active transport across the human erythrocyte membrane. Biochemica of Biophysica Acta, 25: 118-128. Priouzeau, M. 1951. Purpura hemorragique des bovides consecutif'a Z'ingestion de sulphate d'ammoniaque. Rec. Med. Vet., 127: 287-295. Repp, W. W., Hale, W. H., Cheng, E. W., and Burroughs, W. 1955. The influence of oral administration of non-protein nitrogen feeding compounds upon blood ammonia and urea levels in lambs. J. Anim. Sci., 14: 118-131. Rummler, H. J., Laue, W., and Berschneider, F. 1962. Untersuchungen uber die biochemischen vorgange und uber therapeutisehe massnahmen bei der Hainstoffbergiftung der Ringen. Monatsh. Vet. Med., 17: 156—161. 55 Schales, 0., and Schales, S. S. 1941. A simple and-accurate method for the determination of chloride in biological fluids. J. Biol. Chem., 140: 879-884. Schmidt-Nielsen, B., and Osaki, H. 1958. Renal response to changes in metabolism in sheep. Amer. J. Physiol., 193: 657-661. Skeggs, L. T. 1957. An automatic method for calorimetric analysis. Am. J. Clin. Path., 28: 311-322. Somers, M. 1961a. Factors influencing the secretion of nitrogen in sheep saliva. I. The distribution of nitrogen in the mixed and parotid saliva of sheep. Aust. J. Exp. Biol. Med. Sci., 39: 111-122. Stahl, J. 1963. Studies of blood ammonia in liver disease. Ann. Int. Med. ’ 58: 1-240 Summerskill, W. H. J., WOlfe, S. J., and Davidson, C. S. 1947. The metabolism of ammonia and alpha keto acids in liver disease and hepatic coma. J. Clin. Invest., 36: 361-372. Szabo, I. 1966. Prevention of ammonia toxicosis when feeding urea. Acta Agron. Hung., 15: 59-68. Thomas, J. W. 1966. Protein-Kinds and amounts to feed to dairy cattle. Feedstuffs, 30: 58. Topchyan, M. S., and Averyanova, K. G. 1955. Trud. Armyan. Nauchnoissled. Vet. Instr., 8: 179. (Quote from Garner.) Trumper, M. 1929. .Memorandum of'ToxicoZogy. Blakiston, Son and Co., Philadelphia. Turner, T. N. 1967. Urea poisoning in beef cattle. Mod. Vet. Prac., 48: 60. Van Caulaert, C., Deviller, C., and Holff, M. 1932. Le taux de 1' ammoniemie dans certaines affections hepatiques. Comp. Rend. Soc. de Biol., 111: 735. Villarreal, H., Ronces, R., Sanchez, V., and Arcila, H. 1962. Failure of l-arginine to protect in ammonia intoxication. Its role in urea source. Amer. J. Physiol., 202: 364-366. Wahhab, A., Hussain, A., Ali, A., and Muhammad, E. 1955. Utilization of urea in the rations of a cow during maintenance,reproduction and lactation. Pakistan J. Sci. Res., 7: 22-32. Wang, J. H., and Tarr, D. A. 1955. On the mechanism of urease action. J. Am. Chem. Soc., 77: 6205—6206. Warren, K. 1962. Ammonia toxicity and pH. Nature, 195: 47-49. I- .l- w”. 1h"! F E Y" 56 White, L. P., Phear, E. A., Summerskill, W. H. J., and Sherlock, S. 1955. Ammonia tolerance in liver disease: Observations based on catheterization of the hepatic veins. J. Clin. Invest., 34: 158-168. Whitehair, C. K. 1961. A summary of the effects of urea in ruminant rations. E. I. DuPont de Nemours & Co., 18: reprinted from Poultry Feedstuffs. Wilson, R. P., Davis, L. E., Muhrer, M. E., and Bloomfield, R. A. 1968. Toxicologic effects of ammonium carbamate and related compounds. Am. J. Vet. Res., 29: 897-909. Work, S. H., and Henke, L. A. 1939. The value of urea as a protein replacement for dairy cattle. Proc. Am. Soc. Anim. Prod., 32: 404-406. Yoshida, J., Nakame, K., and Nakamura, R. 1957. I. Toxicity of urea and its control. II. Toxicity of ammonium salts and urea in g- rabbits and goats. Jap. J. Zootech. Sci., 28: 185-191. VITA The author was born October 25, 1922, in Denver, Colorado. He attended primary and secondary school in Herington, Kansas, graduating from the latter in 1940. In the fall of 1940, the author enrolled in the preveterinary curriculum at Kansas State University, Manhattan, Kansas, and was accepted into the Veterinary College at the same uni— versity in the fall of 1941 and graduated in the fall of 1944. Following graduation the author worked for Dr. J. J. Lupfer, a general practitioner at Litchfield, Minnesota, until July 1945. In August 1945, he purchased a mixed practice from Dr. 0. Osborn at Paynesville, Minnesota. In February 1949 the author was called into the Armed Forces, Veterinary Corps, with the rank of Lieutenant and served in Chicago, Illinois. He was discharged in February 1950 and returned to Paynesville, Minnesota. The author was recalled into the Armed Forces, Veterinary Corps, in March 1951 with the rank of Captain. Duty stations were Germany and France. He was discharged in December 1952 and entered general practice with Dr. S. D. Sleight at Columbus, Wisconsin. In July 1966, the author aCcepted a position on the faculty at Michigan State University with primary responsibilities to Ambulatory Clinic. At this time he also entered graduate school in the Department of Veterinary Surgery and Medicine at the university. 57