:- i i , ~2- ln‘ 5 . r. s 3:535: TC A??? a? p . ‘V‘ER 532:3 l ‘ 'fi‘ai :2 :31 A ' n‘." i l E ~13. & 1.5,.- I 5: :3. I . . my a .r 3 an... .5. km; a. 5M ‘3. :1 :Mu: .r. u I .. .v- I I “.2. ,w. .5 TH ESl S This is to certify that the thesis entitled The Relationship of Magnesium An: onium Phosphate To F‘roth Production in Runinant Bloat presented by Richard A. Phelps has been accepted towards fulfillment of the requirements for Ph 3 D 0 degree in Dairz w Z/ZM I Mafor pfcéessor Date November 62 1939 LIBRARY Michigan State University THE RELATIONSHIP OF MAGNESIUM AMMONIUM PHOSPHATE TO FROTH PRODUCTION IN RUMINANT BLOAT' By RICHARD A. PHELPS AN ABSTRACT Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Year 1959 Approved ABSTRACT RICHARD A. PHELPS A colorless, crystalline material Spontaneously formed and artificially precipitated from rumen fluid was identified by physical and chemical analyses as mainly hexahydrate magnesium ammonium phOSphate. The hexahydrate magnesium ammonium phOSphate-containing precipitate was quantitatively determined in rumen ingesta obtained from nine rumen-fistulated dairy cows or steers fed a total of 13 nonfroth- or froth-producing rations. The amount of precipitate formed was found to be influenced by the rumen fluid hydrogen ion concentration. The quantity of precipitate formed per unit of hydrogen ions varied with the rations fed. The magnesium and phosphorus intakes of the animals appeared to influence the amount of magnesium ammonium phosphate formed only when a Specific concentration of hydrogen ions was present in rumen fluid. Frothy rumen ingesta exhibited an average of three times more magnesium ammonium phOSphate-containing precipitate and one and four-tenths times more hydrogen ions than nonfrothy ingesta. THE RELATIONSHIP OF MAGNESIUM AMMONIUM PHOSPHATE TO FROTH PRODUCTION IN RUMINANT BLOAT By RICHARD A: PHELPS A THESIS Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1959 C7 2 C 8 (:5 (if. sell :2, ;'{‘ If?" '3 ACKNOWLEDGEMENTS Sincere appreciation is expressed to Dr. C. F. Huffman, Research Professor in Dairy Husbandry, for his edifying guidance and inimitable patience. The author also wishes to thank Dr. N. P. Ralston, Assist- ant Dean of Agriculture, for providing the opportunity to undertake this project. Special thankfulness is extended to Dr. C. K. Smith for his inspiring leadership and cooperation. Gratitude is also expressed to Dr. E. J. Benne, Dr. J. C. Sternberg, Mrs. M. A. Wacasey and Mr. S. T. Bass for aid in chemical analyses, and to L. A. Ried and R. E. Ried for feeding the experimental animals. The writer is also indebted to Mr. Garlon A. Harper, irector, Research and Educational Division, National Cottonseed Products Association, for excellent cooperation during the completion of the thesis. Special thanks are extended to Miss Norma Shosid for typing the manuscript. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . Magnesium Ammonium Phosphate . . . . . . . . . General Properties . . . . . . . . . . . . Natural Occurrence of Struvite . . . . . . Hypomagnesemia and Grass Tetany . . . . . . . . Occurrence of Grass Tetany and Similar Disorders . . . . . . . . . . . . . . . . Hypomagnesemia With Tetany . . . . . . . . Seasonal Trend of Serum Magnesium . . . . Effect of Ration on Hypomagnesemia and Tetany . . . . . . . . . . . . . . . . . . Effect of Pasture Fertilization on Hypomagnesemia and Tetany . . . . . . . . The Relationship of Rumen Ammonia to Hypomagnesemia . . . . . . . . . . . . . Concentration of Other Elements in Blood During Outbreaks of Hypomagnesemia . . . . Herbage Levels of Various Elements During Outbreaks of Hypomagnesemia . . . . . . . Magnesium Content of Forage and Rumen Fluid and Possible Relationship to Bloat . . . . . . . . Rumen Fluid PhOSphate and Possible Relationship to Bloat . . . . . . . . . . . . . . . . . . . Rumen Fluid Ammonia and Possible Relationship to Bloat . . . . . . . . . . . . . . . . . . Rumen Fluid pH and the Possible Relationship to Bloat 0 O I O O O O O O O I O I I O O I O 0 iii Page «PW 0000\1 10 12 13 14 15 I7 20 23 24 . . , . . . . Page EYLIJERII\LEI\TAL P1{OCEDURE o o o o o o o o o o o o o o o o o o o o o 28 Part I. Isolation and Identification of Hexahydrate Magnesium Ammonium Phosphate . . . . . . . . . 28 Part II. Quantitative Measurement of Magnesium Ammonium Phosphate and Hydrogen Ions . . . . . . . . . . . 29 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . 30 Part I. Isolation and Identification of Hexahydrate Magnesium Ammonium Phosphate . . . . . . . . . 30 Part II. Quantitative Measurement of Magnesium Ammonium Phosphate and Hydrogen Ions . . . . . . . . . . . 32 SUl'l-I‘IARY o o o o o o o o o o o o o o o o o o o o o o o o o o o o 4 9 LITELMTURE CITED 0 O O O O O O O O 0 O O O O O O O O 0 O O O O 0 50 iv , -3 .- $3": ’1 7L l Figure I II III IV LIST OF FIGURES Magnesium Ammonium PhosPhate-containing Precipitate Obtained from Rumen Fluid . . . . . . . . . . . . . Relationship of Rumen Fluid Magnesium Ammonium PhOSphate-containing Precipitate to Rumen Fluid Hydrogen Ion Concentration (Multiple Ration Comparisons) . . . . . . . . . . . . . . . . . . Rumen Fluid Magnesium Ammonium Phosphate Precipitate vs. Rumen Fluid Hydrogen Ion Concentration (Single Ration Comparisons) . . . . . . . . . . . . . . . . Rumen Fluid Magnesium Ammonium Phosphate Precipitate vs. Rumen Fluid Hydrogen Ion Concentration (Single Ration Comparisons) . . . . . . . . . . . . . . Rumen Fluid Magnesium Ammonium PhOSphate Precipitate vs. Calculated Magnesium, Phosphorus and Hydrogen in Rumen Ingesta . . . . . . . . . . . . . . . . . . Page 39 41 43 44 45 LIST OF TABLES Table 1 Comparison of Rumen Fluid Crystals with Hexahydrate Magnesium Ammonium Phosphate . . . . . . . . . . . . 2 Ration Components Fed Daily and Rumen Fluid Precipitate Values . . . . . . . . . . . . . . . . . 3 Rumen Fluid pH of Cattle Fed Froth—and Nonfroth- producing Rations . . . . . . . . . . . . . . . . . 4 Calculated Magnesium and Phosphorus Intake, Rumen Fluid Hydrogen Ion Concentration, and Observed Rumen Fluid Magnesium Ammonium Phosphate-containing Precipitate . . . . . . . . . . . . . . . . . . . . 5 Statistical Analyses of Data . . . . . . . . . . . . vi THE RELATIONSHIP OF MAGNESIUM AMMONIUM PHOSPHATE TO FROTH PRODUCTION IN RUMINANT BLOAT INTRODUCTION The riddle of ruminant bloat has plagued husbandrymen for hundreds of years. Until recent times, the incidence of this malady has apparently surpassed research on the etiology of bloat. Recently, however, several practices have been introduced which at least temporarily prevent some forms of bloat. Moreover, additional similarities between the various types of bloat have been conclusively demonstrated. Important in this regard is the indisputable evidence that froth production is an associated factor in cases of both pasture and feed-lot types of ruminant bloat. While this is a significant contribution, the basic cause or causes of this malady have not been infallibly elucidated. The vast amount of research on other cattle disorders may well contribute to an understanding of ruminant bloat. Thus, it is partic- ularly important to study results obtained from research on ”grass tetany", or hypomagnesemia. This malady may occur under conditions of relatively low magnesium intake combined with high ruminal ammonia production. Conversely, ruminant bloat may occur under conditions of relatively high magnesium intake and variable rumen ammonia production. Preliminary data has indicated the possible relationship of an unidentified crystalline material in rumen fluid to froth production. The purpose of this study was to (1) identify the crystalline material, (2) determine the relationship of the material to the hydrogen ion concentration of the rumen, and (3) measure the relative concentration of the crystalline material in the rumen ingesta of cattle fed froth- and nonfroth-producing rations. an? ‘1 n. Danni ’ " REVIEW OF LITERATURE Introduction The role of froth in ruminant bloat has been reviewed or noted by several workers. There has been no previous report of magnesium ammonium phosphate in rumen ingesta, but it has been found in asso- ciation with other bacterial fermentations. This review is therefore concerned with: (1) literature pertaining directly to magnesium am- monium phOSphate and (2) studies bearing upon magnesium, ammonia, phOSphate, and pH in bloated and nonbloated ruminants. Magnesium Ammonium PhOSphate General Properties. Magnesium ammonium phosphate usually exists in the hydrous form with the hexahydrate compound predominating over the monohydrate (Dana, 1884). The hexahydrate form was designated struvite in honor of V. Struve, a Russian statesman, and guanite by Teschemacher (Mellor, 1923). It is a colorless to yellow crystalline compound and may be microscopically identified by immersion in xylene or toluene (Purcell, 1922). Struvite rapidly loses water and ammonia upon heating (Mellor, 1923) and exhibits such properties as tribolumi- nescence (Tomozi, 1939), pyroelectricity and piezoelectricity (Palache, 1945). It exhibits a specific infrared spectrum (Corbridge, 1954). Magnesium ammonium phosphate is formed whenever a neutral magne- sium salt solution is brought in contact with a solution containing ammonia and phOSphoric acid (Mellor, 1923). The compound is soluble in acids and very slightly soluble in water (Dana, 1884). Ammonium oxalate and molybdate exert a greater solubilizing effect than the ammonium salts of hydrochloric, nitric and sulfuric acids or the sodium and potassium salts of hydrochloric and sulfuric acids. However,small quantities of ammonium hydroxide greatly reduce the solubility of struvite (Malyarov, 1934; Uncles, 1946). Magnesium ammonium phosphate inhibits the decomposition of hydro- gen peroxide except when a specific level of cobalt ions is maintained (Krause, 1955). The mechanism of inhibition was not reported. Natural Occurrence g: Struvite. Hexahydrate magnesium ammonium phOSphate was first found in nature in a deposit of cattle manure in Germany (Dana, 1884). It has also been found in a bed of peat under- lying deposits of organic matter (Palache, 1945), in bird and bat guano (Mellor, 1923), in urine (Rodillon, 1914; Cavazzani, 1919; Mellor, 1923; Schwartze, 1923), in extracts from fossil plants (Mellor, 1923), in in- testinal concretions (Mellor, 1923; Hutton, 1945), in prostate glands (Frondel, 1946), in licorice extracts (Jermstad, 1927), with newberyite in the tooth of a Yukon mammoth (Palache, 1945), and in human lungs (Hutton, 1945). Struvite has been numerously reported in urinary cal- culi of several Species including rats, mink and sheep (Milks, 1935; Johnson, 1940; Benjamin, 1945; Sompolinsky, 1950; Vermeulen, 1951; Leoschke, 1952; and Leoschke, 1954) and in human salivary calculi (Bartels, 1950). The urinary calculi in mink appear to be more numer- ous in the females in the spring and in the males in the summer. The calculi are also more numerous among mink in the North Central States than in the Pacific Coast States and may be related to diet. Where the incidence of calculi is high a large prOportion of the mink diet consists of horsemeat (Leoschke, 1952). There is a reported lower in- cidence of urinary calculi on mink ranches feeding high levels of fish (Leoschke, 1954). The formation of magnesium ammonium phosphate urinary calculi in mink can be prevented by the addition to the mink's diet of one gram of ammonium chloride per animal per day (lbid.) The formation of struvite has also been a problem in the canned fish industry (Purcell, 1922; Merwin, 1942; Hollett, 1942; Kreidl, 1951; Dreosti, 1949). Hexahydrate magnesium ammonium phosphate is associated with many organisms. It has been formed around diplococci in urine (Rodillon, 1914); in colonies of Actinomyces (Bartels, 1950; Bartels, 1952) and various unSpecified bacteria (Sierakowski, 1940; Chistyakov, 1952; Barmenkov, 1953); in cultures of Pseudomonas (Fredericq, 1946a; Fredericq, 1946b), Brucella §p, (Huddleson, 1927), and Staphylococcus g2; (Milks, 1935; Kalina, 1952); and associated with staphylococcal or proteus infections (Sompolinsky, 1950). The compound has also been found in cultures of typhoid, paratyphoid and Flexner's dysentery bacillus (Kalina, 1952); and in Bacillus alcaligenes, Neisseria catar- rhalis and Corynebacterium diphtheriae culttres (Scudder, 1928). Magnesium ammonium phosphate has also been reported in samples of tu- berculin and erysipelas antitoxin where formation of the crystals was traced to contaminating ammonia-forming organisms (Babich, 1954). It has also been reported in psychrOphile cultures from Lvov soils; the authors state that the parallelism in seasonal variations of soil phOSphate and psychrOphile cell counts in the soil are evidence that the observed crystallization is part of the nitrogen, phosphorts, and magnesium cycles in plant nutrition (Chistyakov, 1952). Various workers have reported that magnesium ammonium phosphate is the best source of nitrogen tested for the production of soil organisms (Bierema, 1909; Wilson, 1937). The compound has been reported to be effective in complexing ferrous ions to prevent ferrous ion inhibition in the production of penicillin (Ida, 1950). Hypomagnesemia and Grass Tetany The lack of reports concerning magnesium ammonium phOSphate in the rumina of cattle can be attributed to the difficulty in detecting this compound, which would be soluble, and presumably ionic, in the pH range of most rumina. However, many studies of magnesium-ammonia inter- actions, with or without phOSphorus, have been reported with cattle. These studies involve for the most part a hypomagnesemic condition, as in ”grass tetany", ”grass staggers" or "wheat pasture poisoning". Occurrence 9f Grass Tetany and Similar Disorders. Sjollema (1930) reported a high incidence of grass staggers in sandy soil dis- tricts where intensive cattle farming had been practiced. Attacks of the disorder occurred directly after the cows had left the barn, thus excluding grass as the predisposing factor. In 1936, Mulhearn reported that grass tetany cases usually occurred within a month after calving when cows were in heavy milk production. The attacks were reported to coincide, usually, with heat periods and were associated with more or less continuous feeding on rapidly growing roughage. The animals had usually been grazing pasture for two or four weeks before the first cases occurred. It was emphasized that cattle in poor condition are rarely affected. Nolan and Hull (1941) reported several cases of grass tetany when the hypomagnesemic condition occurred during the winter in cattle fed mainly poor-quality hay. Allcroft (1947) reported that only one of 144 cases of hypomagnesemia was associated with turning out to pasture. Kemp and 't Hart (1957) reported that the number of grass tetany cases decreased as the weather became warmer. In general there was little or no further occurrence of grass tetany when the mean 24—hour temperature per decade in the Spring had risen to about 14°C. When the temperature fell below this level in the autumn, there was a further outbreak of the disease. The authors concluded that, consequently, the longer it takes for the temperature to rise above a level of about 14°C, the longer is the duration of the Spring tetany period. They also noted that a high degree of precipitation in the summer and autumn is accompanied by a greater number of tetany cases in these periods. Hyppmagnesemia With Tetany. The reports relating low blood levels of magnesium to the symptoms of tetany have not always been in agreement. Sjollema (1930) reported that both serum and urinary magnesium of cattle were depressed in cases of grass staggers. Nolan and Hull (1941) re- ported 12 cases of hypomagnesemia in cattle accompanied by clinical symptoms of grass tetany. The depression of magnesium was accompanied by a drop in serum inorganic phosphorus. Bartlett gt gl.(l954) reported that all cattle showing tetany in their eXperiment had low serum magne- sium values; however, one cow displayed an abnormally low level of 0.5 to 0.6 mg.% for more than two weeks without displaying tetany. A similar depression of serum magnesium without evidence of tetany has been reported by Allcroft and Green (1938) and Allcroft (1947). Seasonal Trend pf Serum Magnesium. The prevalence of tetany in the spring, and occasionally in other seasons of the year, necessitated a study of the seasonal trend of serum magnesium. Allcroft and Green (1938) reported high values for serum magnesium in the summer and low values in the Spring and winter. The trend was not always evident and clinical symptoms of tetany were more likely to appear when the fall of blood magnesium was sudden. Eveleth gg‘gl. (1940) noted peak values of blood serum magnesium in dairy cattle during the month of July when the animals had been grazing dry pasture grass. The serum magnesium fell when increased rainfall caused a regrowth of the pasture. The occurrence of hypomagnesemic tetany in the winter when cattle were receiving winter rations of poor-quality hay has been reported (Nolan and Hull, 1941). Allcroft (1947) reported that the lowest serum magnesium in cattle ‘I occurred from December to April and the highest level in July, but the finding was not valid with all cattle. The high magnesium values co- incided with periods of low rainfall and little wind while the low values coincided with periods of adverse weather and low temperature. Thus the highest incidence of grass tetany and the lowest values of serum magnesium are apparently coincidental with periods of low tem- perature and adverse weather (Kemp and 't Hart, 1957). Head and Rook (1955) stated that the highest incidence of hypomagnesemia in dairy cattle, as well as decreased urinary magnesium values, occurred in the first few weeks of the Spring grazing which followed the winter stall feeding period. Stewart and Reith (1956a) also reported low magnesium values for cattle in April and progressively higher values from June to July. Bartlett SE g1. (1957b) reported that some cattle showed little or no change in serum magnesium when changed from winter feeding to grazing, irrespective of fertilizer treatment of the swards grazed. In the majority of cattle, however, there was a distinct fall in the serum magnesium, sometimes within 24 hours of the beginning of grazing. If the serum magnesium fall was not acute and the cattle were kept on pasture, there was usually a Spontaneous recovery of the magnesium level after two to three weeks of grazing. The authors concluded that a high serum magnesium level during the period of winter feeding is indicative of a reserve of magnesium in the body which can for a few days prevent the onset of hypomagnesemia. Bartlett and Balch (1957a) reported depressed serum magnesium values during Spring grazing, with some isolated cases of tetany. Effect 2: Ration pp Hypomagnesemia and Tetany. Since not all cases of hypomagnesemia are associated with grazing young herbage, it is important to review alterations of the serum magnesium level by other feeds. Sjollema (1930) reported that hypomagnesemia could re- sult from feeding high-protein rations during winter months. Eveleth g£_§1.(1940) reported that attempts to raise low levels of serum mag- nesium with dry feed and hay were unsuccessful. Noland and Hull (1941), as previously reported, observed grass tetany when cattle were fed a winter ration consisting mainly of poor-quality hay. Allcroft (1947) reported that the feeding of magnesium oxide alleviated the severe fall of serum magnesium which usually occurred when cattle were grazed on Spring pasture. He also reported that the usual winter depression of serum magnesium in cattle was prevented by feeding 15 to 20 pounds of hay and 60 to 90 pounds of cabbage. Allcroft considered the cabbage to be the more important factor since calves which did not receive cabbage did not Show the same rise in serum magnesium. Cabbage has been reported to produce cattle bloat (Johns, 1956). Head and Rook (1955) stated that the magnesium intake during periods of hypomagnesemia associated with Spring grazing is adequate, and that it is generally concluded hypomagnesemia does not arise from an inadequate intake of magnesium. Bartlett _£‘_l. (1957b) reported that supplemen- tation with feeds containing magnesium failed to prevent the onset or reduce the severity of hypomagnesemia. Kemp and 't Hart (1957) reported 11 that the magnesium content of pastures with tetany cases was about ten percent lower than in pastures without cases of tetany. Line (1957) reported that a mixture of two parts linseed cake and one part dredge corn, a mixture of barley, oats, and wheat, prevented the fall of serum magnesium. Rook E£.£l-(1957) observed that young leafy herbage produced a higher incidence of hypomagnesemia than more mature herbage even though the magnesium content was similar. The authors attributed the decreased hypomagnesemia to a greater consumption of the mature herbage. They further observed that the addition of starch to the rumen reduced the severity of hypomagnesemia. Head and Rook (1957) reported a reduction of rumen liquor ammonia following starch addition. Brouwer (1952) noted that grass from tetany-producing pastures was very high in potassium. Kunkel SE El; (1953) reported a decrease of serum magnesium in sheep within 12 days by the inclusion of five percent potassium in the diet. Serum magnesium was also reduced when milo gluten meal replaced soybean meal in the diet. Bartlett £2,2l' (1954) reported that cows grazed on a nitrogen fertilized plot and Supplemented with concentrates did not display the severe fall in serum magnesium Shown by cows not receiving supplementation. None of the concentrate supplemented animals diSplayed clinical symptoms whereas five of eight cows in the nonsupplemented group showed a very rapid fall in serum magnesium, and four of them diSplayed typical tetany symptoms. The fifth animal showed no tetany even though the serum magnesium remained at a low level for more than two weeks. 12 Effect 9f Pasture Fertilization 23 Hypomagnesemia and Tetany. Stewart and Keith (1956) observed that fertilization with ground lime- stone or ground magnesium limestone increased the magnesium content of the herbage and decreased the incidence of hypomagnesemia. However, they stated, ”Thus whilst the magnesium content of herbage was still low there was some degree of recovery to a normal level of blood magnesium. This is further proof that it is not primarily the magne- sium content of the diet which causes the hypomagnesemia or the upset of magnesium metabolism.” Bartlett E£.él; (1957b) concluded that hypomagnesemia in cattle grazing pasture was not dependent on a specific fertilizer application to the pasture. However, they observed the most severe depression of serum magnesium in cattle which had grazed plots dressed with ammonium sulfate. They observed that invariably there was a rise in blood ammonia concentration in cattle which had grazed nitrogen fertilized plots. The extent of the rise was related to the severity of hypomagnesemia except from plots fertilized with magnesite. Bartlett and Balch (1957a) reported that over a period from October, 1956, to September, 1957, cattle grazing a grassland area to which magnesium fertilizer had been applied had a mean blood serum magne- sium level of less than 2 mg.% much of the time. The authors concluded, "Thus, there is as yet no indication that the light dreSsing of magne- sium in the fertilizer has been beneficial in the prevention of hype- magnesaemia.” Bartlett 33 El. (1954) reported that three of four cows on a plot fertilized with a commercial grade of magnesium oxide plus ammonium sulfate showed no hypomagnesemia up to 20 days after the 13 beginning of grazing. The animal that did show low blood magnesium was in no way abnormal in appearance and behavior even though the serum magnesium level decreased to 0.5 mg.%. The serum level was also low in cows grazing a nitrogen fertilized plot; however, the depression was partially prevented if the animals received supplementary concentrates. The lack of tetany in many cases of hypomagnesemia may be partially explained by the studies of Salt (1950). He observed that dairy cattle were the only species of Six studied, including the rabbit, chicken, swine, sheep and human, that yielded higher values of cell than blood serum magnesium. He found a steady fall in corpuscular values following the fall in serum magnesium. The author concluded that this behavior can be best explained by assuming that the level of magnesium in the cell is determined by that of the plasma at the time the erythrocyte is produced in the bone marrow, but that once the cell is cast into the circulation it maintains its mature value unchanged throughout the remainder of its life. In the case of grass tetany the serum mag- nesium falls rapidly, but if the decline occurs over a long period of time the cell magnesium will fall also. The author concluded that the serum and cell magnesium act independently. The Relationship of Rumen Ammonia to Hypomagnesemia. It has been repeatedly Observed that cattle dispky hypomagnesemia after ingesting large amounts of protein (Sjollema, 1930; Mulhearn, 1936; Head and Rook, 1955). Therefore it became important to determine rumen ammonia levels at the time the magnesium disorders occurred. Head and Rook (1955) I c -« u 0 o O V found that the excretion oi magne51um in the urine of the cow is markedly < w- ~~u~——-w-.—= “4‘!“ I, 14 and rapidly reduced with the change to spring grass. The authors pos- tulated that if such a fall in the urinary excretion of magnesium re- flects a reduced intestinal absorption it would indicate that for ruminants the magnesium of Spring grass has an unusually low avail- ability. Head and Rook observed that the level of ammonia in the rumen increased from about 15 mg.% to 50 mg.% when cattle were changed from winter stall feeding to pasture. The high ammonia production from grass was also reflected by the presence of ammonia in the jugular blood, which indicated that the capacity of the liver for conversion of portal blood ammonia had been exceeded. An attempt was made to influence the absorption of magnesium in cattle receiving hay and a concentrate ration by the addition to the rumen, via a fistula, of am- monium acetate or ammonium carbonate. Ruminal ammonia levels com- parable to those observed on grass were produced and a Similar re- duction of urinary and serum magnesium was observed. Rook g£_§l. (1957) later reported that the addition of the ammonium salts decreased urinary excretion of magnesium and increased the excretion of the ele- ment in the feces. Hypomagnesemia could also be produced by adding potassium chloride to the rumen. The authors stated, "These results Show that a high concentration of ammonia or potassium within the rumen leads to an interference with magnesium absorption, and that, when the intake of magnesium is low, this interference leads to the development of hypomagnesemia. Moreover, the change in the pattern of magnesium metabolism following the additions is identical with that observed on changing from winter feed to cut grass.” _§oncentration of Other Elements in Blood During Outbreaks of Hypomagnesemia. The levels of blood constituents other than magne- sium may be altered in outbreaks of hypomagnesemia. Sjollema (1930) - "x.- 15 reported that serum potassium levels were normal but that the calcium: phOSphate ratio was altered. The author stated that the latter abnor- mality may have been due to the high phOSphorus content of the protein- rich feeds. He pointed out that a much higher content of urinary phos- phoric acid had been found in cattle suffering from tetany. Nolan and Hull (1941) reported a terminal drop in the serum inorganic phosphorus from 5.5 mg.% to 3.4 mg.% in 11 cases of hypomagnesemic grass tetany. Allcroft (1947) reported that more than 75% of 144 cases of hypo- magnesemia were associated with hypocalcemia. Bartlett _£__l, (1957b) reported that the most significant change in the composition of the nitrogenous fraction of the blood, following the commencement of graz- ing, was the rise in serum nonprotein nitrogen and urea nitrogen, the latter wholly accounting for the rise of the former. The develOpment of hypomagnesemia resulted in no significant changes in serum concen- trations of amino nitrogen, uric acid, creatinine, protein, calcium, sodium, potassium, glucose or serum pH. Herbage Levels of Various Elements During Outbreaksfigg_Hypo- magnesemia. Kemp and 't Hart (1957) reported that herbage from grass tetany producing pastures had a lower prOportion of magnesium, sodium and calcium, and a higher mean content of potassium and phOSphoruS, than herbage from pastures where no tetany was observed. The ratio of potassium to the other cations was appreciably higher in pastures where tetany resulted. Statistical analyses revealed that a high potassium and low calcium content of the pastures favored the occurrence of grass tetany. No significant correlation was found with the contents of l6 crude protein, magnesium, phosphorus, chlorine and sulfur. However, the magnesium content of pastures with tetany cases was about ten per- cent lower than in pastures without cases of tetany. The authors re- ported a sharp rise of tetany cases as the potassium to calcium plus magnesium ratio (K:Ca1LMg) increased. Since Rook E£.El- (1957) have reported a higher incidence of hypomagnesemia with cattle grazing young rather than more mature herbage from the same plot, it is impor- tant to note the magnesium content of the forage. They found that the content of this element in the forage, the dry matter percentage, and the availability of the element were similar at both stages'of growth. However, the voluntary consumption of the cut mature herbage was some 25% greater, in terms of dry matter. The authors commented, ”The spontaneous and progressive recovery in serum magnesium levels which follows the initial fall at the commencement of grazing may thus be dependent to some extent on an increase in the voluntary consumption of herbage dry matter, and consequently of magnesium, as herbage ma- tures. Any change in the availability of herbage magnesium due to the fall in nitrogen and potassium content of herbage as it matures would also play its part. From results of experiments now completed, it has been concluded that intake of magnesium as determined by the magnesium content and the consumption of herbage, is an important factor in the production of hypomagnesaemia.” Sjollema (1952) attributed grass tetany to deficiencies of sodium and excessive intakes of potassium and nitrogen by cattle. He also attributed tetany to a disturbed metab- olism of phosphorus and cited Norwegian eXperiments that supported his conclusion. Brouwer (1952) observed that grass from tetany-producing, pastures was very high in potassium. He noted that two heavy dressings of a potassium salt not only increased the potassium content of the grass but also decreased the contents of calcium and magnesium. This condition, according to the author, is conducive to the outbreak of grass tetany. Magnesium Content of Forage and Rumen Fluid and the Possible Relationship to Bloat ha: ~r nan-rung... .6 ‘_- Garton (1951) reported the level of soluble magnesium in the ru- men of living fistulated sheep to be 7 to 13 mg.% when fed chOpped meadow hay, and 11 to 20 mg.% when fed a ration of chopped meadow hay, linseed oil cake, and oats. The rumen fluid samples were obtained 2 to 12 hours after feeding. It was observed that the soluble magnesium in the rumen decreased with an increase in time after feeding. The level in the rumina of slaughtered Sheep which had grazed rye grass and clover pastured averaged 6 to 12 mg.%. The soluble magnesium level decreased to 2 to 5 mg.% in slaughtered sheep which had been fed a ra- tion of oats, chopped oat straw, ground corn, and blood meal. Slaugh- tered Sheep which had been fed chopped meadow hay had a soluble magne- sium level of 10 mg.% in the rumen liquor. Carton believed that since the soluble magnesium values found in the ruminal liquor of hay-fed sheep are higher than those reported for saliva, the increase can be accounted for by the Simple solution of these ions from the food. He demonstrated this solution of ions with an ER yiggg experiment. The addition of sheep gastric juice to rumen contents evoked a slight liberation of magnesium from rumen contents. l8 Garner (1949) concluded from ip vitro experiments that ruminal organisms play a significant part in the liberation of magnesium from plant cells. In a later report (1950) he stated that 63 to 75% of the magnesium retained is apparently not dependent upon the magnesium con- tent, the calcium to phosphorus ratio or the crude fiber content of the feed. Lakke Gowda EE El. (1956) reported that with ruminants the amount of magnesium in the ration is directly proportional to the percent hydrolysis of phytate phosphorus. They concluded that the amount of phytate phOSphorus in the ration is the deciding factor in the absorp- tion of magnesium, calcium and phosphorus. Stewart and Moodie (1956b) experimented on anethetized sheep to discover in what parts of the alimentary tract absorption of magnesium takes place. It was shown that after heavy doses of magnesium sulfate under certain conditions, absorption of magnesium may take place from the rumen, abomasum, small intestine and caecum. The authors concluded that the principal Site of magnesium absorption is probably the duodenum and the remainder of the small intestine. After large doses of magnesium salts the phos- phate content of the blood was also increased but the calcium content was slightly decreased. It was shown also that the magnesium was much more quickly and efficiently absorbed after oral doses of magnesium nitrate than after Similar amounts of magnesium sulfate. Legumes, which have been incriminated as an important factor in the production of bloat, generally have a higher content of magnesium than the grasses (Bender and Eisenmenger, 1941; Morrison, 1956). Svanberg and Ekman (1946) reported that a marked deficiency of magne- . 1': _.'_x2£VJ-‘JPF§&‘ ”FT-w v In 19 sium was rarely found on peaty soil, a type of soil reported by Weiss (1953) to yield bloat-producing alfalfa. Svanberg and Ekman (22°El£n) noted that in general forages of low magnesium content were found on acid soils. Similar data was obtained by Bender and Eisenmenger (pp. gi£,) who reported that legumes, with the exception of Sweet clover, contained a higher percentage of magnesium when grown on a basic soil, compared with the same plants grown on a more acid soil. Truog _£._l. (1947) studied the magnesium—phosphorus relationship of peas. They found that the seeds of this legume revealed an appreciable and con- sistent increase in phosphorus content with increasing supplies of available magnesium. In fact increasing Supplies of available magne- sium increased the phosphorus content of the peas much more than did increasing supplies of available phOSphorus. The reduction of rumen fluid pH during outbreaks of ruminant bloat has been reported by Johns (1956, 1957). Thus it is significant that Carr and Woods (1955) reported bovine albumin bound more than nine times as much ionic magnesium at pH 7.5 than at pH 6.0. When the pH was re- duced to 5.5 there was essentially no binding of the ion. Bovine hemo- globin and globulin bound less magnesium than albumin. It has also been reported that magnesium ions were freed from magnesium oleate com— plexes as the pH was reduced to pH 5.7 (Hartsuch, 1938). Albert (1950) studied the binding of amino acids with magnesium ions and found that sometimes the ions gave no evidence of complexing with amino acids. Where positive results were obtained relatively small amounts of magne- sium ions were bound. 20 The production of slime or capsular material by rumen micro- organisms has been Suggested as one factor in the production of frothy bloat (Hungate, 1955; Jacobson, 1957a). Thus it is significant that Stacey (1953) observed the production of large quantities of fructosan and capsules by streptococci grown on a sucrose agar medium containing magnesium. The same organism grown without sucrose and magnesium produced no fructosan and no capsules. Jackson t l. (1957) reported an increase in plasma cholesterol in cattle grazing bloat-producing alfalfa pastures. Moore _£.§l. (1957) reported the production of severe bloating in sheep after ad- ministering a cholesterol-alfalfa juice drench. It is thus noteworthy that Kruse _g._l. (1933) found a marked increase in total blood choles- terol as serum magnesium decreased in dogs fed a low-magnesium ration. There was a commensurate decrease in fatty acids so that total fat re- mained constant. Total cholesterol in ester form was very high. There was a ”terminal rise” in blood nonprotein nitrogen which was explained on the basis of an augmented protein metabolism following failure of the normal fat metabolic cycle. Rumen Fluid Phosphate and Possible Relationship to Bloat Garton (1951) reported a level of 27 to 41 mg.% inorganic phos- phorus in the rumen of fistulated sheep fed a ration of chopped meadow hay. Slaughtered Sheep which had been grazed on rye grass and clover pasture had a level of 42 to 68 mg.% inorganic phosphorus. The feeding of a ration of chOpped meadow hay resulted in a rumen fluid inorganic phosphorus level of 10 mg.% whereas sheep fed a ration of oats, chOpped 21 oat straw, ground corn and blood meal had a very high level of 117 to 164 mg.% inorganic phOSphorus. The author pointed out that ifl.!l££2. experiments revealed a partial precipitation of phosphate with an in- crease in pH from 6.85 to 7.7. Garton remarked that this might happen 12.3333 if rumen contents ever became alkaline. Scarisbrick and Ewer (1951) commented on the high concentration of phosphorus in the rumen. The authors believe that phOSphorus-rich saliva influences the move- ment of phosphorus from the blood to the rumen. They anesthetized sheep, exposed the ruminal vein and carotid artery, and injected into the rumen radioactive phosphorus. The results showed that inorganic phOSphate was absorbed from the rumen in widely varying amounts. On several occasions inorganic phosphate appeared to be recycled from the blood to the rumen. The authors concluded that the net absorption of inorganic phOSphate from the rumen over a long period of time seems to be at most only small in amount. However, at any particular instant there may be a substantial movement of inorganic phOSphate into or out of the blood traversing the ruminal wall. Clark (1953) reported that ruminants actively secreted water-soluble phOSphate in their Saliva. The water-soluble phosphorus in the rumen of sheep fed a phOSphorus- deficient diet was greater than the level in sheep fed a normal diet, even though the opposite was true for the levels in the blood and saliva of the two groups. There was no significant difference between the rumen water-soluble phosphate of cattle grazed on phosphorus-deficient land and those supplemented with bone meal. However, the inorganic phosphorus levels in the blood and saliva were lower in the cattle graz- ing the phosphorus-deficient land. There was no correlation between blood phosphorus figures and the phosphorus content of the rumen. . Fr. ‘1 ' ("u “.3 22 A magnesium-phOSphoruS interaction in ruminants, observed by Lakke Gowda and co-workers (1955), has been reported previously. They con- cluded that the percent hydrolysis of phytate phosphorus seems to be directly preportional to the amount of magnesium present in the ration, and that the amount of phytate phosphorus in the ration is a deciding factor in the absorption of magnesium as well as calcium and phosphorus. The possible relationship of phosphorus to bloat has been advanced in several reports. Troughton (1955) stated that most pasture bloat from white clover was produced on soils poor in soluble phosphorus pentoxide and potassium oxide. The difference in bloat potential of clover blocks was not attributed to herbage production. The relation- ship did not hold true when plots within a block were compared. The author pointed out that this may have been due to overshadowing by dif- ferences due to mixtures. Cooper and Hall (1956) reported that bloat was eSpecially common in areas where soils were deficient in available phOSphorus. The authors stated, ”In bloat, a rich nitrogen or protein ration seems to be one of the significant predisposing factors. Only rarely does bloat occur from the grazing of leguminous crOps produced on soils which have received liberal applications of phosphorus, or on soils where there is a geological accumulation of available phOSphoruS in the soil.” The authors thought that if the nitrogen to phosphorus ratio changes from about 11 to 1 to approximately 15 to 1 there is a potential bloat hazard. They state that where there is a relatively large excess of nitrogen in plants, unstable azo, azoxy and diazo organic groupings may become significant factors in determining the accumulation of gases in the rumen. COOper and Woodle (1957) claimed 23 that fatal bloat cases were reduced five to eight times by increased phosphorus fertilization on land grazed by several hundred head of cattle. Since various workers have implicated bacterial slime as a factor in bloat (Hungate, 1955; Jacobson, 1957a), the findings of Rorem (1955) appear noteworthy. This worker reported that cells associated with a slime polysaccharide covering had an uptake of phOSphate ions 2 to 20 times that of the same number of cells without slime or capsules. The author stated that the results of this study seemed compatible with the hypothesis of polysaccharides being an important factor in ion binding by bacterial and higher plant cells. Rumen Fluid Ammonia and Possible Relationship to Bloat The reduction of magnesium in the blood and urine of cattle fol- lowing the ingestion of high-protein rations has prompted workers to study the possible role of increased rumen ammonia in this reduction. Lewis g£_§l. (1957) Showed that there was a correlation between rumen and portal blood ammonia over a wide range of rumen ammonia concen- trations. Johns (1955) observed high rumen ammonia levels in sheep after they had grazed high-protein herbage. The range of concen- tration was from 35 to 130 mg.% of ammonia nitrogen. The peak levels did not coincide with the peak protein levels in the herbage, nor did the low concentrations necessarily result from the ingestion of low protein herbage. The pH of the rumen fluid was never above 6.5. It has been reported previously that Head and Rook (1955) observed higher rumen ammonia values for grazing cattle than for cattle fed a dry ration. 24 Briggs SE El. (1957) reported that the volatile fatty acid levels in the rumen are adequate for the neutralization of large amounts of ammonia. However, they state, ”Nevertheless it appears certain that the pH-volatile fatty acid relationship is modified to some consid- erable degree by high ammonia nitrogen levels.” They found that the rumen ammonia levels were highest at high levels of volatile fatty acids. I— The possible relationship of high levels of ammonia to ruminant bloat has been suggested by Hale and King (1955). They believed that g the conditions in the rumen following urea-induced bloat are Similar to those observed when ruminants suffer from legume pasture bloat. Brown g£_§l. (1957) reported a positive correlation between bloat severity and ammonia content of rumen ingesta, but reduction of bloat by oils or an antibiotic did not reduce rumen ammonia values. Johns (1955) found that high ammonia levels can be maintained in the rumen of sheep fed winter grass without the appearance of any signs of bloat or toxicity. Jamieson and Loftus (1958) reported the sporadic presence of a long filamentous bacterium of the Beggiatocaceae family in the rumina of sheep on pasture. They observed that the presence of them was indicative of a sustained level of ammonia nitrogen lower than the 11 to 18 mg.% usually found in rumen liquor of pastured cattle. Rumen Fluid pH and the Possible Relationship to Bloat Johns (1956) has recently shown that the hydrogen ion concentra- tion in the rumen may differ between bloated and nonbloated cattle. He noted that the pH of the rumen was always below 6.5 when cattle bloated an. r 3 "736 Mg}. .2; '- . . .' ‘ -. - 25 on pasture. Johns _£_§l.(l957) cited unpublished work of Reid which confirmed that the rumen fluid was in the region of pH 6.3 at the on- set of bloat, and as rumen fermentation proceeded the pH dropped below 6.0. Johns (1956) cited unpublished work of Mangan who found that in the presence of salts red clover cytoplasmic proteins at a level of 20 mg.% protein nitrogen formed a very stable foam, with a maximum stability at pH 6.0. Johns g£_§l. (1957) observed that the maximum foam strength of the rumen liquid occurred when the liquid was in the region of pH 6 with a salt concentration of 0.15 to 0.2 molar. A determination of the Optimum pH for maximum foam stability for red clover saponins and cyto- plasmic protein extracts revealed that while the former is about pH 5, that of the latter is approximately the same as the rumen contents, or pH 6. The authors concluded that with this evidence it appeared that at the pH of the rumen contents at which bloat occurs protein is likely to be more important as a foaming agent than the saponins. They further pointed out that plant protein was rapidly released in the rumen. The level of protein necessary to produce a stable foam accumulated within 30 minutes after the animals had been placed on pasture, the length of time in which animals have been observed to bloat. The interaction of bovine proteins, pH, and magnesium, reported by Carr and Woods (1955), was previously mentioned. They found that bovine serum albumin bound more than nine times as much ionic magnesium at pH 7.5 than at pH 6. When the pH was reduced to 5.5 no magnesium was bound by the bovine albumin. It was found that bovine globulin bound less than albumin. These observations may partially explain the influence of pH and salt concentration on foam stability reported by Johns (1956). 26 Since cattle have been reported to exhibit stable froth formation after ingesting immature alfalfa or Ladino clover plants (Weiss, 1953; Boda t 1., l956; Nichols, 1956; Johns, 1958) as well as with feed lot bloat (Smith t al., 1953; Phelps, 1956; Jacobson, 1957b) it is noteworthy that Ammerman and Thomas (1952) reported that juice from nonbloating blue grass forage was more highly buffered than were juices from such bloat-producing forages as Ladino clover and alfalfa. They also found that juices from more mature Ladino and alfalfa appeared to be more highly buffered than juices from the same forages when young and succulent. his is a significant finding when one considers that cattle appear to bloat more on young succulent legumes than on either mature legume or grass pastures. The authors further reported that the rumen ingesta pH of lambs grazing alfalfa or Ladino clover averaged approximately pH 6.24. When the lambs were grazing bluegrass pasture the pH rose to 6.56. Following prefeeding of hay, a practice sometimes utilized to prevent bloat, the rumen ingesta pH increased to 6.7 with legume pasture grazing and 6.9 with grass pasture grazing.' Monroe and Perkins (1939) reported a somewhat reversed observation. They observed a rumen ingesta pH of 6.47 when the cattle were grazing bluegrass pasture and a pH of 6.66 when the animals grazed alfalfa. Cason and others (1954) fed various legume and grass roughages and found that the highest rumen fluid pH coincided with the least weight of wet material in the rumen, the lowest percentage of dry matter, and the highest ash content. A high degree of relationship was found to exist between the ash content of the ingesta and the pH of the rumen. 27 The authors concluded that the ash moved out of the rumen more slowly than the other constituents of the ingesta and exerted a buffering action on the rumen fluid, thus influencing rumen fluid pH. ' EXP ER [MEN TAL P ROCEDU RE This study consisted of two parts with Part I composed of isola- tion and identification of hexahydrate magnesium ammonium phosphate. Part 11 involved quantitative measurements of the mineral complex and hydrogen ion concentration in rumen fluid. Part 1. Isolation and Identification of Hexahydrate Magnesium Ammonium Phosphate juduil‘m'I "(3 I' Rumen ingesta was obtained from mature, rumen-fistulated dairy steers or cows which had been fed a froth-producing ration develOped by Smith gt El. (1953). The ingesta was strained through four layers of cheesecloth and the fluid portion centrifuged at a relative cen- trifugal force of 30,000 for 15 minutes in a Servall SS-l centrifuge. Aliquots of the supernatant obtained were titrated to each 0.5 of a pH unit between pH 7 and 13 with 10% NH4OH. Titration to a value of pH 9.0 was found to yield the largest amount of pure precipitate. The precipitate was permitted to stand for several hours and then redissolved in dilute HCl. The resulting solution was filtered through Whatman No. 1 filter paper and the filtrate obtained titrated to pH 9.0 with 10% NH4OH. Crystallization was permitted for several hours after which the material was separated by decantation. The crystals were air-dried and then vacuum-dried for 29 hours over con- centrated H2504. Qualitative analyses were carried out spectro- graphically. Elements shown to be present by these analyses, as well as ash and nitrogen, were determined quantitatively. 28 29 Part II. Quantitative Measurement of Magnesium Ammonium Phosphate and Hydrogen Ions Rumen ingesta for magnesium ammonium phosphate determinations was obtained from nine rumen-fistulated dairy cows or steers ranging in age from one to seven years. A total of 13 nonfroth and froth-producing rations was fed. Rumen fluid pH determinations were carried out with eight animals fed a total of six nonfroth and froth-producing rations. Rumen ingesta was obtained ventro-medial to the fistula and immedi- ately prior to 8 A.M. feeding except when hourly samples were obtained. The ingesta was strained through four layers of cheesecloth and hydrogen ion determinations were immediately performed on the strained rumen fluid by means of a Beckman Model H-2 potentiometer equipped with calomel and glass electrodes. The strained fluid was then removed to the laboratory and centrifuged at a relative centrifugal force of 700 for five minutes in an International centrifuge. The supernatant obtained was then cen- trifuged at a relative centrifugal force of 30,000 for ten minutes in a Servall SS-l centrifuge. The clear fluid obtained with divided into 25- or 50-ml. aliquots and adjusted to pH 9.0 with 10% NH4OH. The base was added from a burette, and a Beckman H-2 potentiometer was employed to measure pH values. The rumen fluid was immediately poured into 50-ml. volumetric flasks with care being taken to transfer all of the formed precipitate. A lU-ml. calibrated Kolmer Pyrex centrifuge tube was then placed over the neck of the flask and the flask inverted, causing the material in the flask to move to the tip of the tube. The vacuum created inside the inverted volumetric flask prevented any loss of rumen fluid. The quantity of rumen fluid precipitate was recorded as soon as precipitation was complete. . 11'“; -v‘ 0 . V _ 9 RESULTS AND DISCUSSION Part I. Isolation and Identification of Hexahydrate Magnesium Ammonium Phosphate The spectrographic analysis of rumen fluid crystals revealed the presence of large amounts of phosphorus and magnesium plus a trace of ‘ calcium. The results of the ash and elemental analyses of the crystals and the calculated values for hexahydrate magnesium ammonium phosphate (MONH4PO4°6H20)~are presented in Table 1. Table 1. Comparison of Rumen Fluid Crystals with MgNH4PO4-6H20 Compound Percentage Ratio of N;Mg;P Ash N Mg P Crystals from Rumen Fluid 47.14 5.90 10.07 12.77 1:1:1 MgNH4P04-6H20 45.35 5.71 9.91 12.63 1:1:1 Confirmation of the presence of MgNH4PO4-6H20 was obtained by infrared spectrosc0py. The presence of MgNH4PO4°6H20 crystals spontaneously formed in centrifuged rumen fluid was confirmed by the microsc0pic method of Wherry (cited by Purcell and Hickey, 1922). This method involved the microsc0pic observation of MgNH4PO4‘6H20 crystals suspended in toluene, a liquid having the same refractive index as the crystals. As MgNH4P04°6H20 crystals are brought into and out of focus, the bands of light along the edges of the crystals alternate between yellow and blue. Several precautions must be observed in analyzing for rumen fluid MgNH4P04°6H205. Carbon dioxide is continually released from the ingesta with a resulting pH increase. Centrifugation of the ingesta must be 30 31 completed before the pH has risen to approximately pH 7 or spontaneous crystallization will occur with the resulting loss of the product. The Optimum pH for precipitation of MgNH4P04.6H20 from centrifuged rumen fluid is pH 9.0. If higher pH values are employed, rumen fluid pigments will be precipitated with :he mineral complex. Precipitation may be carried out below pH 9.0, but the product yield will be lowered. Acidification is commonly employed in the determination of NgNH4P04.6H20 by precipitation. During the development of an analytical procedure for this mineral complex, it was found that the addition of HCl to rumen fluid would usually increase the amount of precipitated MgNH4P04.6H20. However, it was important to measure only the NgNH4P04.6H20 that would form from the addition of NH4OH to material soluble in the rumen fluid. If acidification were employed, a further release of com- plexing material from previously unavailable sources would be expected to occur. Garner (1949) has demonstrated by an ig_yi££2 technique that more ultrafilterable Mg appeared as simulated ruminal contents underwent increased acidification. Precaution must be observed in drying the MgNH4P04.6H20 crystals. All water of hydration is lost if the drying temperature is allowed to reach 100 degrees centigrade. High temperatures also result in loss of ammonia. It was found that drying over sulfuric acid in a vacuum jar gave satisfactory results. Water of hydration greatly influences the infrared spectrum of MgNH4P04.6H20. Therefore, it is necessary to treat the known sample of the mineral complex in exactly the same manner as the unknown. It was found that infrared Spectra of the known and unknown compounds 32 could be matched by dissolving the compounds in dilute HCl, precip- itating the material by addition of 10% NH4OH to pH 9.0 and drying the obtained precipitate over concentrated H2804 for 29 hours. Part II. Quantitative Measurement of Magnesium Ammonium Phosphate and Hydrogen Ions Identification of the rumen fluid crystals from frothy rumen in- y gesta was followed by a study of the comparative amounts in frothy and nonfrothy rumen ingesta obtained from animals fed a variety of rations. It was known that the method for measuring MgNH4P04 was accurate for different aliquots of the ggmg rumen fluid sample; in the second phase, the study included a comparison of the accuracy of the method between rumen fluid samples. The values for MgNH4P04-containing precipitate, hereafter desig- nated MgNH4P04, formed from rumen fluid of dairy cattle fed a variety of rations are shown in Figure I. The values are eXpressed as the vol- ume per 100 m1. of rumen fluid and per m1. of added base; thus, the quantity of added ammonium ions is equalized. The rations employed in the study are shown in Table 2 together with the range ofvalues obtained for MgNH4PO4. The data presented in Figure I reveal that more precipitate was obtained from those animals displaying frothy rumen ingesta than those with nonfrothy except for one animal, No. 24. The relatively large amount of MgNH4P04 obtained from this animal displaying nonfrothy in- gesta occurred when the animal was "off feed" and exhibiting very acidic rumen fluid. The effect of this highly acidic ingeshaon the yield of 33 MgNH4P04 is discussed below. It is important to point out that’frothy rumen ingesta yielded an average of three times more MgNH4P04 than non- frothy ingesta. It is significant that identical twin animals, 25 and 26, fed identical rations, did not reSpond similarly. Twin 25 had previously been fed a high-roughage ration for several weeks before receiving the daily ration of 10 lbs. of concentrate No. 24 and 10 lbs. of alfalfa hay. This animal did not diSplay frothy rumen ingesta. The twin of this animal, 26, although fed an identical ration, did diSplay frothy L_ ingesta. However, this animal had previously been receiving a froth- producing, high-concentrate ration for several weeks. It is note- worthy that the animal diSplaying the frothy ingesta also yielded rumen fluid with a greater concentration of both MgNH4PO4 and hydrogen ions. It is significant that cattle fed rations containing a large per- centage of roughage produced rumen fluid containing low levels of MgNH4P04. The rumen fluid samples from these animals usually exhibited a much higher pH value than rumen fluid obtained from cattle fed a ration containing a large percentage of concentrates. Statistical analyses of 115 rumen fluid samples revealed a highly significant correlation between the amount of MgNH4P04 formed and the amount of base added to rumen fluid obtained from nine animals fed a total of 11 rations. However, the correlation between the same two factors was not significant with data obtained from animals fed high levels of roughage. This lack of correlation was believed due to the inaccuracy of the method when measuring extremely small amounts of MgNH4P04 in rumen fluid obtained from roughage-fed cattle. 34 In Figure II, it can be seen that the hydrogen ion concentration of the rumen fluid, as measured at the moment of sampling, does not appear to be correlated with the amount of MgNH4P04 obtained from the rumen fluid. It must be emphasized that a comparison is being made between eight different animals fed six different rations, and thus there may be a correlation between the amount of MgNH4P04 formed from and hydrogen ion concentration of rumen fluid obtained from single animals fed single rations. This correlation will be discussed below. 1 Figure II also illustrates that a given ration may result in a relatively constant amount of MgNH4P04, even though measured in dif- ferent animals, if the rumen pH values are similar. Thus, rumen fluid from animals 25 and 26 yielded the same high amount of the mineral complex when the animals were fed ration G, a mixture of alfalfa hay and corn-soybean oil meal concentrate. Likewise, animals 23 and 139 yielded similar low amounts of MgNH4P04 when fed identical rations of timothy hay, ration M. Similarly, data presented in Figure I shows that animals 3 and 25, fed approximately equal amounts of alfalfa hay, ration K, yielded almost equal amounts of MgNHAPOA. Thus, the amount of MgNH4P04 obtained from rumen fluid appears to be influenced by the composition of the ration fed. It has not been ascertained whether the ration components exert a direct effect on the MgNH4PO4 formed or whether the effect results indirectly from altered rumen metabolism. However, the latter explanation appears more probable as will be discussed below. 'In addition to the data shown in Figure II, Table 3 contains data obtained from hourly sampling of rumen fluid from four cattle fed four 35 different rations. The data illustrate the difference in rate of for- mation of the mineral complex when different rations are fed. The results also show the parallelism between the hydrogen ion concentra- tion of rumen fluid and the amount of MgNH4P04 formed from rumen fluid when comparisons are made on the basis of individual rations. Since the greatest amount of MgNH4P04 was usually observed in rumen fluid obtained five hours after feeding, it appeared that the amount of the complex formed may be related to the production of one or more of the volatile fatty acids. Moir and Somers (1957) reported that the total volatile fatty acid production reached a peak approximately four hours after feeding. Similar data have been reported by Reid _E._l. (1957) who also noted that the production of prOpionic acid paralleled the production of total volatile fatty acids. Butyric and acetic acid did not usually follow the same trend as prOpionic acid. Thus, it appears that the production of one or more volatile fatty acids may influence the amount of MgNH4PO4 formed. Since only one of the rumen fluid samples in Table 3 was from frothy ingesta, no comparison can be made between froth production and resulting MgNH4P04 concentration of rumen ingesta. It is note- worthy,however, that the average amount of MgNH4POhformed over the seven-hour period was directly proportional to the calculated mag- nesium and phosphorus intakes of the animals. Additional data illustrating the relationship between rumen fluid hydrogen ion concentration and MgNH4P04 are shown in Figures III and IV. Again, the parallelism between the concentrations of hydrogen ions and MgNH4P04 is evident in almost all cases. Statistical analyses of 36 the data revealed this correlation to be significant to the one percent level of probability with animals 25 and 26, to the two percent level with animal 2, and to the five percent level with animals 24 and 130. Data from the three remaining animals showed less statistical correla- tion of the above two factors as shown in Table 5. These animals were the only ones of the group receiving an all-roughage ration. The amount ".1 of MgNH4PO4 formed when these rations were fed was extremely low, and thus lack of statistical significance can be explained by the inability m‘.fm-O\l of the method to accurately measure small amounts of MgNH4P04. A lack of correlation between rumen fluid hydrogen ion concentra- tion and MgNH4PO4 was pointed out in the discussion of Figure II, whereas in the discussion of Figuresllflland IV and Table 3 a correla- tion was shown to exist for five of the eight animals. The results are not conflictiHO. The correlations in Figure II are based on com- parisons between different rations. However, all correlations between rumen fluid hydrogen ion concentration and MgNH4PO4 in Table 3 and Figures III and IV are based on data obtained from the feeding of a single ration. Thus, the comparison in the latter case is not between vastly different rations as in the former case. Therefore, it appears that the rumen fluid hydrogen ion concentration, as well as the compo- sition of the ration fed, greatly influences the amount of MgNH4P04 formed in rumen fluid. The Specific reason why hydrogen ion concentration should effect the yield of MgNH4PO4 obtained from rumen fluid is unknown. However, the previously cited work by Garner (1949, 1950) illustrated the in- crease in magnesium ions as rumen ingesta was acidified. Similarly, 37 Carr and Woods (1955) observed a decreased binding of Mg ions by bovine proteins with increasing concentration of hydrogen ions. A similar observation was made by Hartsuch (1938) who found that Mg was released from oleate complexes when the pH was decreased. If the release of both Mg and P04 ions increases with greater acidification of rumen fluid, a partial explanation for the increased amounts of MgNH4PO4 in 3f .4"; 18“! rumen fluid treated with NHAOH is offered. The amount of mineral ions released in rumen fluid of a constant pH will be different for different EAL}? 3‘; ‘ ,, s rations; thus, a feasible explanation is offered for the experimentally determined differences in MgNH4P04 obtained from rumen fluid of cattle fed different rations. An attempt to correlate the combined effect of the Mg and P in- take plus rumen fluid hydrogen ion concentration with the determined amount of MgNH4PO4 is shown in Figure V and Table 4. The values ob- tained for the Mg and P content of the ration components were taken from the mineral analyses of feeds published by Morrison (1956). The hydrogen ion concentration was obtained directly from the rumen as re- ported in the experimental procedure. Figure V illustrates the results of the comparison and the formula employedto obtain the values. It can be readily seen that there is a direct relationship between the combined factors of Mg and P; or Mg, P, and H ions; versus the determined amount of MgNH4P04-containing precipitate in only half of the comparisons. Even though the method of comparison is arbitrary, the values are still relative since all comparisons were made by means of the same formula. 38 Previously, it was shown that data presented in Table 3 indicated a direct relationship between calculated Mg and P intake and MgNH4P04 formation. Again, there is no conflict of data. The comparison of results shown in Table 3 is between the average amount of MgNH4P04 obtained over a seven-hour period versus the calculated intake of Mg and P. The data illustrated in Figure V compare the same calculated l' i intake of Mg and P with average concentrations of MgNH4PO4 obtained prior to feeding. In the former comparison, MgNH4P04 values were 1 obtained when considerable rumen volatile fatty acid production was occurring. The effect of this acid production and the resulting rumen pH depression can have a profound effect upon the mineral ions present in the rumen as was discussed above. Thus, it appears that the amount of MgNH4PO4 formed in rumen fluid may be influenced by the Mg and P intake of the animal if the rumen ingesta becomes sufficiently acid. In conclusion, it must be emphasized that the amount of MgNH4P04 obtained from rumen fluid appears to be dependent both on the rumen fluid hydrogen ion concentration and on the ration fed. The average amount of MgNH4P04 obtained from frothy rumen ingesta was three times more than that from nonfrothy ingesta. However, it must be pointed out that cattle fed rations capable of producing low rumen pH values may produce nonfrothy rumen ingesta yielding highMgNH4PO4 values. Lower rumen pH values, however, do appear to be associated with both froth production, as shown in this study when identical twin cattle were fed identical rations, and pasture bloat as reported by Johns (1956) and Johns t g1. (1957). Figure I. MgNHaPOA-containing Precipitate Obtained from Rumen Fluid Animal Ration Frothy Rumen In esta 2 A.IIiiIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII'IIIIIII' 2 B— 21. c— 2 n— 1 e— 24 F— 26 c— Nonfrothy Rumen Ingesta--Animal Not Eating Well 24 F — Nonfrothy Rumen In esta 130 H b 25 G — 130 I — 23 J — 25 K _ 26 L- 3 K— 23 M- 139 )1. Average of all Frothy Ingesta Samples Average of all Nonfrothy Ingesta Samples Table 2. Ration Components Fed Daily (lbs.) and MgNH4P04 Values . _ (All Rations Include 50 g. NaCl Daily) _ [Data corresponds to ration and animal designation shown on opposite pagg/ MgNHggohgg Alf. Conc. Conc. Conc. Tim. Other No. Range of’ Hgy_ #24* #25** #52*** Hay Components Samples Values Mean (ml./lOO ml.rumen fluid) 6 8 11 .33-3.04 1.12 o 8 800 ml.9SZEtOH 4 .75-l.64 1.11 4 12-14 4 .42-l.53 .97 Ration 3 minus alcohol 5 .31-l.38 .85 Ration A plus 30 ml. Citroflex 7 .22-1.ll .83 4 0-7 ”-14 9 .30-1.52 .74 10 u) 14 .43-l.41 .71 Same as Ration F above 4 .43-1.94 .95 12 2 lb.No. 2 corn, ground 9 .14-1.51 .56 Same as Ration C above 15 ,17-1,10 .49 Ration H plus 100 mg. Procaine Penicillin lo .27-0.76 .42 4 8-10 o .l8-O.60 .41 20-30 10 .l3-0.63 .34 Ration K plus 400 m1. 957 Ethanol 8 .08-0.63 .32 Same as Ration K above 23 ,07—0,58 .30 11-20 17 0-0.27 .09 Same as Ration V above 1% U-U,28 v3 32 .22-3.04 an in} 0-1.9§ 31 Concentrate Rations I lngreifi'Corn,Corn andtf Corn Narcol Trace dient ,Croundpogb Meal 30f! Starch 8-73 l’re:',(‘.;.C(‘_'LCaE?PQ/,L Sa1tjs’it..:‘x‘.'it.1) Ration 24777.0 20.0 1.0 1.0770,} 5310P4.5x103¥ Ration 25 69.5 20.0 8.0 1.0 1.” 0.5 5x10F415x105 Ration 52 77.7 19.4 2.9 . 41 m1. MgNH4PO4/100 ml. rumen fluid/ml. added base 0 guru-v Fig. II. Relationship of Rumen Fluid MgNHaPOg-containing Precipitate to Rumen Fluid Hydrogen Ion Concentration (Multiple Ration Comparisons) Hlx 107 - 2 0 26-G* 25-6 2-A 130-1 24-F 24-F 3-K 23-M '139-M Fr Fr Fr '(Fr ' Frothy Ingesta) Animal and Ration *Refer to Table 2 for ration composition Table 3. Rumen Fluid pH and MgNH P04 producrng Rations of Cattle Fed Froth and Nonfroth- m1.MgNH4P04/ 100ml. rumen Sampling No. Rumen Fluid_pH Mean H fluid/m1. Animal Ration Time* Samples Range Mean x10'7 added base 26** C 0 4 6.4-7.1 6.6 2.6 0.59 25 G 0 5 6.6-7.2 6.9 1.3 0.59 2** A O 3 6.6-6.8 6.7 2.0 0.49 130 '1 0 5 6.5-6.9 6.7 1.9 0.48 24 F 0 4 6.2-6.6 6.5 3.5 0.41 24*= F 0 l 6.7 6 7 2.2 0.36 3 k 0 5 7.1-7.3 7 2 0.6 0.23 - 23 M 0 5 6 6-7.2 6 9 1.4 0.06 139 M 0 5 6.5-6.8 o 7 1.8 0.04 2 A 0 2 6.4-6.5 6.5 3.5 0.88 2 A 2 2 6.1-6.2 6.1 7.5 3.26 2 A 5 2 5.2-5.8 5.4 35.5 6.37 2 A 7 2 5.8 5.8 15.9 5.44 3 K 0 2 7 1 7.1 0.8 .32 3 K 2 2 6.5-6 7 6.6 2.6 1.33 3 K 5 2 5.9-6.0 5.9 11.6 7.58 3 K 7 2 5.9-6 1 6.0 10. 8.11 23 J o 2 6.5-6.9 6.6 2.3 .35 23 J 2 2 6.3-6.7 6.5 3.3 .54 23 J 5 2 5.6-6.0 5.7 19.1 1.15 23 J 7 2 5.6-6.0 5.8 17.1 .96 24 F 0 2 6.4-6.6 6.5 3.0 .58 24 F 2 2 5.6-6.0 5.7 19.6 2.19 24 F 5 2 . 5 2 5.2 61.0 1.76 24 F 7 2 5.3-5.4 5.4 44.9 -- Av. of all frothy ingesta samples 52 5.8-7.2 o 5 3.4 .90 Av. of all nonfrothy ingesta samples 105 5.7-7.6 6.6 2.5 .31 *Hrs. after feeding **Frothy Rumen Ingesta ***Portable potentiometer used on series samples 42 11.. ammo m.~ o.~ ¢.a ~.H xxa-oS x ma.cv 1r Aa-oa x w~.a-e us cm x A TE x 3.8 i. 3.2 x R; n» N x hm-ca x «H.~v 3r Am-ca x No.-v H» mm x ATS x 3.33 i- ATS x a; 2» am mm 43 cuppa .fiexvasae coast .a5 cea\eonq=zwz .45 o.~ w. c. Q. N. ’ O _ O ‘. ...t... .. V1 .02 deem—E n .92 3:554 0 .2. 3...... D. D .67. Amec< . > -— 6N Aumvom Aumv N wcwvomm ucwcuoe ea begun ado .«owEEw cmcwmuno mwnmm> ~_< Anaemwuwascu comumm odwcmmv coauouucoucou cow cowouvhn viadw smash .a> acquzwz candy cease HHH .mfim vo.¢ .o.m rh4 L01 X 44 x,Ak-ea x qa.quxe-oa x ma.-v x xa-oa x am.mVirmno~ x oo.~v x A “-3a x mm.vnrAa-o~ x mm.v x A “-2s x wmroerAa-oH x ca.v H H II >- >- >- >< Amcomflpoaeuoo oojwm oawcnmv cowumuucoocoo co... cowouoxs was: smash .m> 3&6:sz ofifim coaam >H .wfim «was sauna .as\nasau coast .He cea\ somezzmz .aa omfioz deems,» . mmadz HmEHE<< m .02 A935» 0 mm .02 Hmeac< .v .N.« o.a w o o o *0 N o . , . . 1)-- 1 .1.-- I and i . Q _. Amv nun“ . .6 A. . I I . .. .- d. m q i can .v 4+ 2 mewnmmw mewcpos o Momma 3336255 665320 magma» :< OJ o.~ o.m 01v o.m ., H) £01 XL; f, "j II 45 Fig. V. Rumen Fluid MgNH4P04 vs. Calculated Mg, P, and H in Rumen Ingesta 0.8. = Rumen fluid MgNH4P04 (m1./100 m1. rumen f1uid/m1.added base) = ”43* x 10) -/- (1b.P* x 10) -/- (3/1 H”** x 106) 3 0.7- —"= swimmer: 2'10.) 2 e : calculated daily intake of element ** 3 measured hydrogen ion concentration in rumen ; ->< x x-u.-x-x*>t,x x >.< x x x ><'"x x 'x x x xx xx x xxx x xii-1 'xxx xxx} wikax x x. x x x x x x “xi: x x xxx-"x1 {30: xx x'.x,.x3)¢ ‘xxxgx x x £4 L_ .'.:J-ga . 1 ~ i" .x xxx x x- first-x as I] ,3, xxx x xxx. .x xix-1 "I N‘ U) U F! 93' v ‘0 130 Animal Numbers mm Table 4. Calculated Mg and P Intake, Rumen Fluid Hydrogen Ion Concentration, and Observed Rumen Fluid Magnesium Ammonium Phosphate-containing Precipitate Values used in Calculations Ingredient % Mg Corn & Cob meal 0.085’l SOM 0.27' Alfalfa hay 0.31 Timothy hay 0.17 Ration 24* 0.13 ‘Ground Corn 0.10 Corn starch 0,10% Ration 52* 0.085 Dicalcium phosphate 0 #value estimated from whole grain value Frothy Rumen Fluid Daily Intake(lb.) Animal Ingesta Ration lflfig/lx10’7 Mg____ P 25 1L G 2.02 0.044 0.058 25 - G 1.25 0.044 0.058 2 7L A 2.02 0.029 0.042 130 - I 1.86 . 0.039 0.034 23 - J 8.14 0.020 0.032 24 - F 3.50 0.024 0.040 3 -— K 0.62 0.093 0.072 139 - M 1.85 0.034 0.028 *Composition given in Table 3 **x = (1b.Mg X 10)}(1b.P x 10)%(g/1 Hlfx 109) 3 ***y 3 (lb.Mg x 10)/(lb.P x 10) 2 46 % P 0.22 0.64 0.24 0.14 0.34 0.27 0.27’‘ 0.22 0.18 nfl.MgNH4P04/ 100ml . rumen fluid/ml. x** y*** added base 0.43 0.51 0.59 0.38 0.51 0.59 0.30 0.35 0.49 0.30 0.36 0.48 0.44 0.26 0.44 0.33 0.32 0.41 0.57 0.82 0.23 0.27 0.31 0.04 ' '—1§n' . 1‘ r.- w—z» awn-v“ «wt-Warm; Table 5. Statistical Animal Ration* No. Samples X** Y*** r8 tb pc MgNHAPOA-ppt. (X) vs. Hydrogen Ion Concentration (Y); Animal & Ration Comparisons 2 A 4 .78 3.02 +.987 +8.70 (.02 25 c 5 .59 1.25 +.988 +8.85 (.01 28 c 5 .52 2.24 +.983 +8.19 ( .01 130’ I 5 .48 1.88 +.907 +3.73 (.05 24 F 5 .41 3.30 +.879 +3.19 (.05 3 K 5 .23 0.83 +.888 +1.84 >.05 23 n 5 .08 1.38 +.558 +1.18 ).05 139 M 5 .04 1.85 —-.183 — .32 >.05 MgNHaPoa-ppt. (X) vs. 1 2 3 23 24 25 26 130 139 14 15 16 12 15 14 16 .73 .62 .12 .05 .27 .17 .21 .21 .04 .73 .70 .41 .39 .59 .41 .54 .49 .40 47 +.987 +.570 0 —.243 +.253 +.184 +.249 -.302 4-10.82 4 .01 +2.40 (.05 0 — .94 + .83 + .87 + .89 -l.19 m1. of added 101 NH40H (Y); Animal Comparisons >.OS >.OS ).OS ).05 ).OS >.05 ).05 Analyses of Results Ration* Animal No. Samples X“ yeee MgNHAPOA-ppt. ml. MgNH4P04/100 m1. Rumen Fluid/m1. added base A 2 3 B 2 4 C 24 5 D 2 7 E 1 5 F 24 7 C 25,26 11 H 130 9 I 130 7 K 3,25,26 24 L 26 7 M 23,139 21 A11 Samples 115 4 Refer to Table 2 4 ':=': 3.11% 107/1. Rumen Fluid a : b I t 2 rV N - 2 V7???- c : .34 .84 .65 .61 .73 .29 .29 .20 .13 .13 O [0 £\ 48 .69 .78 .67 .66 .73 .65 .49 .44 .43 .48 .40 0 —-.646 +.908 +.858 54.987 14.482 +.293 -.430 0 +.179 mL.101 -.186 4.034 Coefficient of Correlation :b/WL- 52y.xfl S Y P 2 Probability or Level of significance X) vs. m1. of added 102 NHAOH (Y); Ration Comparisons 0 >.05 —1.19 >.05 +3.70<.05 +3.70(.02 +10.82 (.01 +1.17 >.05 + .92 >.05 —1.26 >.05 0 >.05 + .85 >.05 + .37 >.05 — .82 >.05 +8.69<.01 SUMMARY The isolation and identification of a colorless, crystalline compound Spontaneously formed and experimentally precipitated from rumen fluid was found to be mainly hexahydrate magnesium ammonium phOSphate. The amount of this material precipitated from rumen fluid was influenced by the hydrogen ion concentration of the rumen fluid. The quantity of material formed was related to the type of ration fed. However, animals fed identical rations yielded amounts of magnesium ammonium phOSphate proportional to rumen fluid hydrogen ion concentration. The magnesium and phOSphorus intakes of the animals appeared to influence the amount of magnesium ammonium phosphate formed only when a specific concentrationcf hydrogen ions was present in rumen fluid. The amount of magnesium ammonium phOSphate-containing precipitate isolated from rumen-fistulated animals displaying frothy ingesta was approximately three times that of animals exhibiting nonfrothy ingesta. 49 LITERATURE CITED Albert, A. 1950. Quantitative Studies of the Avidity of Naturally Occurring Substances for Trace Metals. 1. Amino-Acids Having Only Two Ionizing Groups. Biochem. J. 47:531-538. Allcroft, W. M., and H. H. Green. 1938. Seasonal Hypomagnesaemia of the Bovine, Without Clinical Symptoms. J. Comp. Pathol. Therap. 51:176-191. Allcroft, W. M. 1947. Seasonal Hypomagnesaemia of the Bovine Without Clinical Symptons. Vet. J. 103:75-100 .-‘—3 Ammerman, Clarence B., and W. F. Thomas. 1952. Variations in the 4— Buffering Capacity of Rumen Juice as Affected by the Ration. J. Animal Sci. 11:754-755. Babich, M. A., and V. A. Plotnikova. 1954. The Nature of Crystals Forming in Byproducts. Zhur. Mikrobiol., Epidemio. lmmunobiol. No. 2, 11-12. (Chem. Abstr. 48:9017, 1954). Barmenkov, Ya. P. and N. V. Sokk. 1953. The Chemical Nature of Crystals Formed During the Cultivation of Certain Pathogenic Microorganisms. Trudy Chkalov. Sel' skokhoz. Inst. 6:105- 113. (Chem. Abstr. 50:5140, 1956). Bartels, Henry A. 1950. Bacterial Growth and Crystal Formation: A Possible Factor in Calculus Formation. J. Dental Research 29: 436-439. (Chem. Abstr. 44:10883, 1950). Bartels, Henry A. 1952. Microorganisms in Salivary Calculus Forma- tion. N. Y. State Dental J. 18:241-248. (Biol. Abstr. 27:1581, 1953). Bartlett, 8., B. B. Brown, A. S. Foot, and S. J. Rowland, Ruth Allcroft, and W. H. Parr. 1954. The Influence of Fertiliser Treatment of Grassland on the Incidence of Hypomagnesaemia in Milking Cows. Brit. Vet. J. 110:3-19. Bartlett, 8., and C. C. Balch. 1957a. Hypomagnesaemia and Grass Tetany. Natl. Inst. Research Dairying (Reading, England), Rept. 1957, p. 45. 50 51 Bartlett, 8., B. B. Brown, A. 5. Foot, M. J. Head, C. Line, J. A. F. Rook, S. J. Rowland, and G. Zundel. 1957b. Field Investigations into Hypomagnesaemia in Dairy Cattle. With Particular Reference to Changes in the Concentration of Blood Constituents During the Early Grazing Period. J. Agr. Sci. 49:291-300. Bender, William H., and Walter S. Eisenmenger. 1941. Intake of Certain Elements by Calciphilic and Calciphobic Plants Grown on Soils Differing in pH. Soil Sci. 52:297-308. Benjamin, John A., James G. Wilson, and Alice D. Leahy. 1945. Experi- mental Urinary Calculi. 11. Quantitative Microchemical, Spectro- graphic, and Citric Acid Analyses of Albino Rat Calculi, With a Preliminary Apatite Report. J. Urol. 54:516-524. Bierema, S. 1909. The Assimilation of Ammoniacal, Nitrate, and Amid Nitrogen by Microorganisms. (Abs.) Chem. Soc. 96:692-693. (EXpt. Sta. Record 21:620, 1909). Boda, J. M., P. T. Cupps, Harry Colvin, Jr., and H. H. Cole. 1956. The Sequence of Events Preceding Death of a Cow in Acute Experimental Bloat on Fresh Alfalfa TOps. J. Am. Vet. Med. Assoc. 128:532-535. briggs, P. H., J. P. Hogan, and R. L. Reid. 1957. The Effect of Volatile Fatty Acids, Lactic Acid, and Ammonia on Rumen pH in Sheep. Australian J. Agri. Research 8:674-690. Brouwer, E. 1952. On the Base Excess, the Alkali Alkalinity, the Alkaline Earth Alkalinity and the Mineral Ratio in Grass and Hay with Reference to Grass Tetany and Other Disorders in Cattle. Brit. Vet. J. 108:123-131. Brown, L. R., R. H. Johnson, R. S. Allen, N. L. Jacobson, and P. G. Homeyer. 1957. The Relationship of Plant Composition, Rumen Fluid Characteristics and Weather to the Incidence and Severity of Bloat. J. Animal Sci. 16:1083. Carr, Charles W., and Kenneth R. Woods. 1955. Studies on the Binding of Small Ions in Protein Solutions With the Use of Membrane Electrodes. V. The Binding of Magnesium Ions in Solutions of Various Proteins. Arch. Biochem. BiOphys. 55:1-8. Cason, James L., E. S. Ruby, and O. T. Stallcup. 1954. The Influence of the Ash Content of the Rumen Ingesta on the Hydrogen Ion Concentration in the Bovine Rumen. J. Nutrition 52:457-465. Cavazzani, E. 1919. Crystals of Ammonium Magnesium PhOSphate in Star- Form in Urine and Their Value in the Preliminary Study of Magnesium Exchange. Riforma med. 34, No. 20; Arch. ital. biol. 69:80-96. (Chem. Abstr. 14:762, 1920). 52 Chistyakov, F. M., and 2. F. Kamneva. 1952. Crystallization in cultures of PsychrOphiles. Mikrobiologiya 21:540-547. (Chem. Abstr. 47:7035, 1953). Clark, R. 1953. A Study of the Water Soluble PhOSphate Concentra- tion of the Ruminal Contents in Normal and PhOSphorus Deficient Animals. Onderstepoort J. Vet. Research 26:137-140. Cooper, H. P., and E. E. Hall. 1956. Significance of the Coequal Effect or Balanced-Baulic-Poundage Ratio of Major Fertilizer Nutrients to Relative Percentage Growth ReSponse in Cr0p Plants. Soil Sci. 82:201-216. Cooper, H. P., and H. A. Woodle. 1957. Balanced Fertilization f? Controls Bloat. Plant Food Rev. 3:7-9. ' Corbridge, D. E. C., and E. J. Lowe. 1954. The Infrared Spectra of L Some Inorganic PhOSphorus Compounds. J. Chem. Soc. Part I, . 493-502, 1954. Dana, James D. 1884. Manual of Mineralogy and Lithology, John Wiley and Sons, Inc., New York, N. Y., p. 231. Dreosti, G. M., and R. P. Van der Merwe. 1949. Struvite in Canned Crawfish. Fish Ind. Research Inst., Cape Town, South Africa, Progr. Rept. No. 9. (Food Sci. Abstr. 23:443, 1951). Eveleth, M. W., D. F. Eveleth, and F. E. Walsh. 1940. Hyper- magnesemia Without Clinical Symptoms in Dairy Cattle. J. Dairy Sci. 23:85-89. Fredericq, P. 1946a. Formation of Crystals in Bacterial Cultures, Compt. rend. soc. biol. 140:792-795. (Chem. Abstr. 41:4827, 1947). Fredericq, P. 1946b. Factors Determining the Appearance of Crystals of Magnesium Ammonium Phosphate in Bacterial Cultures. Compt. rend. soc. biol. 140:795-797. (Chem. Abstr. 41:4827, 1947). Frondel, Clifford, and Edwin L. Prien. 1946. Deposition of Calcium PhOSphates Accompanying Senile Degeneration and Disease. Science 103:326. Garner, R. J. 1949. Availability of the Magnesium of Grass to the Ruminant. Nature 164:458. Garner, R. J. 1950. Availability of the Magnesium of Grass to the Ruminant. Nature 166:614. Garton, G. A. 1951. Observations on the Distribution of Inorganic PhoSphorus, Soluble Calcium and Soluble Magnesium in the Stomach of the Sheep. J. Exptl. Biol. 28:358-368. 53 Hale, W. H., and R. P. Ring. 1955. Possible Mechanism of Urea Toxicity in Ruminants. Proc. Soc. Exptl. Biol. Med. 89:112-114. Hartsuch, Paul J. 1938. The Chemical Reaction Between Oleic Acid and Aqueous Solutions of Magnesium. Its Pathologic Significance. Arch. Path. 25:17-23. Head, M. J., and J. A. F. Rook. 1955. Hypomagnesaemia in Dairy Cattle and Its Possible Relationship to Ruminal Ammonia Production. Nature 176:262-263. Head, M. J., and J. A. F. Rook. 1957. Some Effects of Spring Grass on Rumen Digestion and the Metabolism of the Dairy Cow. Proc. Nutrition Soc. (England and Scotland) 16:25-30. Hollett, Andrew. 1943. Magnesium Ammonium Phosphate Crystal Forma- tion in Canned Lobster. J. Fisheries Research Board Can. 6: 183-193. (Chem. Abstr. 37:6754, 1943). Huddleson, I. Forrest, and O. B. Winter. 1927. Magnesium Ammonium Phosphate Crystals in Aerobic Cultures of Brucella abortus and Brucella melitensis. J. Infectious Diseases, 40:476-478. Hungate, R. E., D. W. Fletcher, R. W. Dougherty, and B. F. Barrentine. 1955. Microbial Activity in the Bovine Rumen: Its Measurement and Relation to Bloat. Appl. Microbiol. 3:161-173. Hutton, C. Osborne. 1945. The Nature of an Enterolith. New Zealand J. Sci. Technol. 26Bz304-307. (Chem. Abstr. 39:5320, 1945). Ida, Michio, and Miyazawa Homare. 1950. Studies on the Media for Penicillin Production. TV. A Probable Mechanism of the Stabil- izing Action of Penicillin Fermentation Liquor on Penicillin Against the Destroying Effect of Heavy Metal Ions. I. The Case of Ferrous Ion in Aqueous Solution. J. Antibiotics (Japan) 3: 717-720. (Biol. Abstr. 28:887, 1954). Jackson, H. D., R. A. Shaw, W. R. Prichard, and B. W. Hatchet. 1957. Alterations in Blood Constituents of Cattle During Bloat. J. Animal Sci. 16:1047. Jacobson, Don R., Ivan L. Lindahl, J. J. McNeill, J. C. Shaw, R. N. Doetsch, and R. E. Davis. 1957a. Feedlot Bloat Studies. 11. Physical Factors Involved in the Etiology of Frothy Bloat. J. Animal Sci. 16:515-524. Jacobson, D. R., L. D. Brown, D. R. Dowden, F. H. Baker, and R. B. Grainger. 1957b. Excessive Stable Froth Formation in the Reticulo-Rumen as the Primary Cause of Legume Bloat. J. Dairy Sci. 40:615-616. 54 Jamieson, N. D., and T. M. Loftus. 1958. A Characteristic Filamentous Bacterial Rumen Organism, Indicative of a Sustained Low Ruminal Ammonia-Nitrogen Level. 1958. New Zealand J. Agr. Research 1: 17-30. Jermstad, A. 1927. Presence of Magnesium Ammonium PhOSphate in Com- mercial Licorice Extract. Norg. Apotekerforen Tidsskr. (9); Quart. J. Pharm. 1:121-122. (Chem. Abstr. 23:2533, 1929). Johns, A. T. 1955. Pasture Quality and Ruminant Digestion. II. Levels of Volatile Acids and Ammonia in the Rumen of Sheep on a High-Production Pasture. New Zealand J. Sci. Technol. A 37:323-331. Johns, A. T. 1956. Bloat. Vet. Revs. Annot. 21107-134. Johns, A. T., J. L. Mangan, and C. S. W. Reid. 1957. Bloat. New Zealand Vet. J. 5:115-118. Johns, A. T., J. L. Mangan, and C. S. W. Reid. 1958. Animal Factors in the Aetiology of Bloat. Proc. New Zealand Soc. Animal Prod. 18:21-30. Johnson, D. W., L. S. Palmer, and J. W. Nelson. 1940. Failure of Dietary Magnesium Imbalance to Produce Urinary Calculi in Wethers. Vet. Med. 35:353-357. Kalina, G. P., and B. A. Kikhman. 1952. Crystallization in Bacterial Cultures. Mikrobiologiya 21:528-539. (Chem. Abstr. 47:7035), 1953. Kemp, A., and M. L. 't Hart. 1957. Grass Tetany in Grazing Milking Cows. Neth. J. Agr. Sci. 5:4-17. Krause, Alfons. 1955. Specificity of PhOSphates as Carriers of Catalysts in Oxidation-reduction Systems. Roczniki Chem. 26:165-172. (Chem. Abstr. 49:13752, 1955). Kreidl, Ekkehard L., and Earl P. McFee. 1951. Preventing Formation of Struvite in Canned Cooked Fish and Shellfish. Chem. Abstr. 45:6319 Kruse, H. D., Elsa R. Orent, and E. V. McCollum. 1933. Studies on Magnesium Deficiency in Animals. 111. Chemical Changes in the Blood Following Magnesium Deprivation. J. Biol. Chem. C:603- 643. Runkel, H. 0., K. H. Burns, and Bennie J. Camp. 1953. A Study of Sheep Fed High Levels of Potassium Biocarbonate with Particular Reference to Induced Hypomagnesemia. J. Animal Sci. 12:451—458. 55 Lakke Gowda, H. S., N. D. Kehar, and N. K. Ayyar. 1955. Studies on Phytic Acid Phosphorus Metabolism in Ruminants. 1. Distribu- tion of Phytic-acid Phosphorus and Total PhOSphorus in Some of the Common Indian Cattle Feeds. 2. Influence of High, Low and Medium Levels of Ingestion of Phytic-acid Phosphorus on Calcium, Phosphorus, and Magnesium Metabolism. Indian J. Med. Research 43:609-616. (Nutrition Abstr. & Revs. 26:519, 1956). Leoschke, W. L., E. Zikria, and C. A. Elvehjem. 1952. Composition of Urinary Calculi from Mink. Proc. Soc. Exptl. Biol. Med. 80:291- 293. Leoschke, W. L., and C. A. Elvehjem. 1954. Prevention of Urinary P‘ Calculi Formation in Mink by Alteration of Urinary pH. Proc. Soc. EXptl. Biol. Med. 85:42-44. Lewis, D., K. J. Hill, and E. F. Annison. 1957. Studies on the Portal Blood of Sheep. 1. Absorption of Ammonia from the Rumen of the Sheep. Biochem. J. 66:587-592. Lindahl, Ivan L., R. E. Davis, Don R. Jacobson, and J. C. Shaw. 1957. Feedlot Bloat Studies. 1. Animal and Dietary Factors. J. Animal Sci. 16:165-178. Line, C. 1957. Grass Tetany; The Value of Supplemental Feeding When Turning Out to Pasture. Natl. Inst. Research Dairying (Reading, England), Rept. 1957, pp. 36-37. halyarov, K. L., and V. B. Matskievich. 1934. Solubility of Mag- nesium Ammonium Phosphate in Certain Salt Solutions. Z. anal. Chem. 98:31-33. (Chem. Abstr. 28:6361, 1934). 1ellor, J. W. 1923. A Comprehensive Treatise ggpInogganic and Theo- retrical Chemistry. Longmans, Green and Co., London, pp. 384-387. Merwin, R. T. 1942. Struvite in Canned Fish Products. Conn. Agr. Expt. Sta. Bull. 460, pp. 429-430. Milks, H. J. 1935. Urinary Calculi. Cornell Vet. 25:153-164. Moir, R. J., and M. Somers. 1957. Ruminal Flora Studies. VIII. The Influence of Rate and Method of Feeding a Ration Upon Its Digesti- bility, Upon Ruminal Function, and Upon the Ruminal Population. Australian J. Agri. Research.8:253-265. Monroe, C. F., and A. E. Perkins. 1939. A Study of the pH Values of the Ingesta of the Bovine Rumen. J. Dairy Sci. 22:983-991. Moore, C. L., V. A. Hall, and A. E. Dracy. 1957. Bloat. Results From Various Drenchings, Including Effectiveness of Penicillin for Prevention. 'J. Dairy Sci. 40:616. Morrison, F. B. 1956. Feeds and Feeding, 22nd Ed., The Morrison Publishing Co., Ithaca, N. Y., pp. 1096-1099. Mulhearn, C. R. 1936. Grass Tetany. Queensland Agri. J. Advisory Leaflet No. 18. Nichols, R. F. 1956. Excessive Frothing in the Rumen Produced by Fresh Legume Tops. J. Am. Vet. Med. Assoc. 128:215. Nolan, A. F., and F. E. Hull. 1941. Grass Tetany in Cattle. Am. J. Vet. Research 2:41-45. Palache, Charles, Harry Berman, and Clifford Frondel. 1945. The System pf Mineralogy of James Dwight Dana and Edward Salisbugy Dana. 7th Ed. Vol. II, John Wiley and Sons, Inc., New York, N. Y., pp. 715-717. Phelps, Richard A. 1956. A Study gf Some Chemical and Physical Fac- tors gf Rumen Fluid §§_Re1ated £9 Frothy Bloat 33 Cattle. M.S. Thesis, Library, Michigan State University, E. Lansing. Purcell, C. S., and C. H. Hickey. 1922. Note on the Occurrence of Struvite in Canned Shrimps. Analyst 47:16-18. Reid, R. L., J. P. Hogan, and P. K. Briggs. 1957. The Effect of Diet on Individual Volatile Fatty Acids in the Rumen of Sheep, With Particular Reference to the Effect of Low Rumen pH and Adaptation on High-starch Diets. Australian J. Agri. Research 8:691-710. Rodillon, G. 1914. Twinned Crystals of Magnesium Ammonium Phosphate in Urinary Sediments. Bull. Sci. pharmacolog. 20:527-531. (Chem. Abstr. 8:735, 1914). Rook, J. A. F., M. J. Head, M. Wood, and S. J. Rowland. 1957. Hypo- magnesaemia and Grass Tetany. Natl. Inst. Research Dairying (Reading, England), Rept. 1957. pp. 78-79. Rorem, Edward S. 1955. Uptake of Rubidium and Phosphate Ions by Poly- saccharide-producing Bacteria. J. Bacteriol. 70:691-701. Salt, Frederick John. 1950. Magnesium in Cells and Plasma, With Particular Reference to Bovine Blood. The Lab. J. 8:357-367. Scarisbrick, R., and T. K. Ewer. 1951. The Absorption of Inorganic PhOSphate From the Rumen of Sheep. Biochem. J. 49:LXXIX. Schwartze, E. W. 1923. Triple (Magnesium Ammonium) Phosphate Forma- tion and Cystitis From Fethanol. J. Pharmacol. 21: Proc. 216- 217. (Chem. Abstr. 17:3546, 1923). Scudder, Sara A. 1928. The Precipitation of Magnesium Ammonium Phos- phate Crystals During the Growth of Bacteria in Media Containing Nitrogenous Substances. J. Bacteriol. 16:157-161. 57 Sierakowski, Stanislaus. 1940. Formation of Crystals in Bacterial Cultures and the Possibility of Obtaining Secondary Cultures From These Crystals. Compt. rend. soc. biol. 134:64-66. (Chem. Abstr. 36:510, 1942). Sjollema, B. 1930. On the Nature and Therapy of Grass Staggers. Vet. Record 10:425-430. Sjollema, B. 1952. Over de Oorzaken en Gevolgen van Irrationele Opname van Macro-elementen bij Melkkoeien en de Behoefte van Melkkoeien aan Deze Elementen. Nationale Cooperative aan-en VerKOOp-vereniging voor de Landbouw, Central Bureau, G. A. Rotterdam. “a Smith. C. R., J. R. Brunner, C. F. Huffman, and C. W. Duncan. 1953. Experimental Production of Frothy Bloat in Cattle. J. Animal Sci. 12:932. _ Sompolinsky, David. 1950. Urolithiasis in Mink. Cornell Vet. 40: 367-377. Stacey, M. 1953. Bacterial Polysaccharides. Endeavour 12:38-42. Stewart, J., and J. W. S. Reith. 1956a. The Effects of Magnesian Liming on the Magnesium Content of Pasture and the Blood Level of Magnesium in Cows. J. Comp. Pathol. Therap. 66:1-9. Stewart, J., and E. W. Moodie. 1956b. The Absorption of Magnesium From the Alimentary Tract of Sheep. J. Comp. Pathol. Therap. 66:10-21. Svanberg, 0., and R. Ekman. 1946. Om Magnesium—halten i Vegetationen Fran Svenska Jordar. Kgl Lantbruksakad. Tidskr. 85:54-99. (Nutrition Abstr. & Revs. 16:548, 1946-47). Tomozi, Inoue, Minoru Kunitomi, and Eiiti Sibata. 1939. Triboluminescence. J. Chem. Soc. Japan 60:149-156. (Chem. Abstr. 33:4522, 1939). Troughton, Arthur. 1955. A Comparison of Three Strains of White Clover. II. Observations on Bloat. J. Brit. Grassland Soc. 10:297-305. Truog, Emil, R. J. Goates, G. C. Gerloff, and K. C. Berger. 1947. Magnesium-phosphorus Relationships in Plant Nutrition. Soil Sci. 63:19-25. Uncles, R. F., and G. B. L. Smith. 1946. Solubility of Magnesium Ammonium PhOSphate Hexahydrate. Ind. Eng. Chem., Anal. Ed. 18:699-702. 58 Vermeulen, C. W., R. Goetz, H. D. Ragins, and W. J. Grove. 1951. Ex- perimental Urolithiasis. IV. Prevention of Magnesium Ammonium Phosphate Calculi by Reducing the Magnesium Intake or by Feeding an Aluminum Gel. J. Urol. 66:6-11. Weiss, K. E. 1953. The Significance of Reflex Salivation in Relation to Froth Formation and Acute Bloat in Ruminants. Onderstepoort J. Vet. Research 26:241-250. Wilson, J. K. 1937. The Production of Macroscopic Colonies on Plaques of Soil. J. Am. Soc. Agron. 29:286-292. —“-- ‘ ianfi- ’J _ .1 ,1; ,5 Q i la; 7" “'5‘ ‘3: if T' p “’7‘ *3 -" “Me-18 "‘71, Q. 76 ‘1": H H LIIIII'I Y”9 I” 3|! R“ E“ V" N”! U" 03174 4 ll 3 1293 1111111111111