A STUDY OF SOME CHEMICAL AND PHYSICAL PROPERTIES OF RUMEN FLUID AS RELATED TO FROTHY BLOAT IN CATTLE Thais far The Degree of M. S. MICHIGAN STATE UNIVERSITY Richard A. Pheips W56 (a x‘n A STUDY OF SOME CHEMICAL AND PHYSICAL PROPERTIES OF RUMEN FLUID AS RELATED TO FROTHY BLOAT IN CATTLE By RICHARD A. gfiELPs AN ABSTRACT Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for thc degree of MASTER OF SCIENCE Department of Dairy Year 1 956 Approved r p . u m a.— QI . 3L. _1 . s ' . r If r IC 1.- VI"; U I 1 i .z‘ a Q,E J IPSTRACT RICHARD A. PHELPS Rumon fluid samples from two fistulated dairy steers vero suhjected to various chemical and physical determinations. The anirals were fed one non—froth producing and two froth producing rations to ascertain the oossihle relationship of frothy rumen fluid to frothy bloat. The results of this study shofied that there was wide variahility of rumen fluid nitrogen fractions hetween animals on the same ration. There was also considerable daily vari— ation of rumen fluid nitrogen. Total soluble rumen fluid nitrogen £33 se did not appear to influence froth production. The determination of nonprotein and protein nitrogen of rumen fluid by trichloroacetic acid precipitation did not appear to be a valid method due to incomplete precipitation of the proteins. Heat coagulable and non-heat coagulable rumen fluid nitrogen values, when expressed as a percentage of the total soluble nitrogen, were extremely variable between both rations and animals, and they could not be correlated with froth production. Rumen fluid nonprotein nitrogen as determined by an alcohol precipitation method, isplayed considerable corre- lation with frothing, when it was expressed as a percentage of the total soluble nitrogen. Rumen fluid protein nitrogen, as determin:d by an r.) XVSTRRCT RICHARD A. PHELPS Rumon fluid samples from two fistulatec dairy steers were subjected to various chemical and physical de-teerminat1ions. The animals were fed one non—froth producing and two froth ble relationship of l-Jo producing rations to asce ain the pose frothy rnmen fluid o frothy bloat. The results of this study shofied that there was wide variability of rumen fluid nitrogen fractions between animals on the same ration. There was also considerable daily vari- ation of rumen fluid nitrogen. Total soluble rumen fluid nitrogen per se did not appear to influence froth production. The determination of nonprotein and protein nitrogen of rumen fluid by trichloroac tic acid precipitation did not appear to be a valid method due to incomplete precipitation of the proteins. Heat coagulable and non-heat coagulable rumen fluid nitrogen values, when expressed as a percentége of the total soluble nitrogen, were extremely variable between both rations and animals, and they could not be correlated with :.roth produ ucti on. Rumen fluid nonprotein nitrogen as determined by an alcohol precipitation method, displayed considerable corre- lation with frothing, when it was expressed as a percentage of the total soluble nitrogen. Rumen fluid protein nitrogen, as determined by an APS RACT RICHARD A. PHELPS alcohol precipitation method, did .‘ show a positive rela- : Q (. tionship to frothing. The flow time of rumen fluid did not appear to be correlated with froth production. Rumen fluid from the non-froth producing ration was found to yield a more stable foam than in the case of froth producing rations. Rumen fluid from the non-froth producing ration also yielded the greater column of foam when air was bubbled through the liquid. A STUDY OF SOME CHEMICAL AND PHYSICAL PROPERTIES OF RUMEN FLUID AS RELATED TO FROTHY BLOAT IN CATTLE -- Ry 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 MASTER OF SCIENCE Department of Dairy 1 956 3358-28 r“ ' 5 V‘T‘l _‘, ‘J ‘,’ L.\ [.1 \J . .IJ ',‘_- - Ant.‘ - Ana‘. 0 'I d 1 Q ‘ ’11:.qu aIJ' 1"”"0'" r73."7r-,‘F«“\"t"~‘ eta.-.” 3114.4 \1.u.Jll..J.". L-‘ The writer wishes to eXpress his sinc re appreciation to Dr. C. F. Huffman, Research Professor ’3 Dairy Husbandr“ d ’ for his inspiration, cooperation, ad"' the preparation of this manuscript. Tee author also wishes to thank Dr. Earl weaver for providing the opportunity to undertake this task. Gratitude is also eXpressed to Dr. J. Robert Brunner, Dr. Clyde K.Smith, and Dr. George M. Ward for advice and criticism; to Dr. .arold M. Sell for analysis of rumen ingesta foam; to R. H. Crampton, T. W. Crampton, J. F. Long, I. P. Fisher, and R. J. Rothhaupt for laboratory assis- tance, and to L. A. Ried and R. E. Ried for feeding the eXper- imental animals. The writer also wishes to thank Mrs. L. w. Specht for typing the manuscript. ii q .. “u 4 3 TI. D s JV ..PI.. Mud Tl R “- ‘9‘ . -m—w TABLE OF CONTENTS INTRODUCTION REVIEW 0? LITERATURE Introduction . . . . . . . . . . . . . . . . Physiology of Bloat . . . . . . . . . . . . . Types of Bloat . . . . . . . . . . . Factors Influencing Bloat . . . . . . . Physical deficiency . . . . . . . . . . Excessive gas . . . . . . . . . . . . . Toxic gas . . . . . . . . . . . . . . . Flavones and unknown forage extracts Saponins . . . . . . . . . . . . Hydrocyanic acid Ammonium carhamate . . . . Histamines and allergies . . . . . . . . Saliva . Buoyancy . . . . . . . . . . . . . Abnormal rumen flora . . . . Minerals and soil factors Heredity . . . . . Anatomical defects Preventive Measures . . . . . . . . Prior feeding of hay . . . . Soiling . . . . . . . . . . . Alternate grazing of grass and legume pastures Intermittent versus continuous grazing Proper grass-legume ratio Gra7ing mature plants iii Page o; LU L» h‘ '33 “\1 \l u‘\ 1 '6 Page All sgmenys . . . . . . . . . . . . . . . . ’i? Feeding antifrothing compounds . . . . . . 4E Feeding minerals . . . . . . . . . . . . . 43 Regulating water consumption . . . . . . . 43 Correcting anatomical abnormalities . . . 44 Treatments for Float . . . . . .5. . . . . . . 44 Turpentine or kerosene . . . . . . . . . . 44 Formaldehyde . . . . . . . . . . . . . . . 45 Lipase treated cream . . . . . . . . . . . 45 Silicone compounds . . . . . . . . . . . . 45 Household detergents . . . . . . . . . . . 46 Adrenalin and atropine sulfate . . . . . . 46 Mechanical treatments . . . . . . . . . . 46 EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . 47 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 51 Total Soluble Nitrogen . . . . . . . . . . . . 51 Nonprotein Nitrogen from Trichloroacetic Acid Treatment . . . . . . . . . . . . . . . . 5: Protein Nitrogen from Trichloroacetic Acid Treatment . . . . . . . . . 53 Non-heat Coagulahle Nitrogen 54 Heat Coagulahle Nitrogen 55 Nonprotein Nitrogen by Alcohol ' Precipitation . . . . . . . . . . . . . . . . . 56 Protein Nitrogen by Alcohol Precipitation . . . 57 Flow Time . . . . . . . . . . . . . . . . . . . 58 Foam Stability and Foam Height . . . . . . . . 59 SUMMARY . . 78 LITERATURE CITED . . . . . . . . . . . . . . . . . . 80 iv Figure II III III IV IV VI VI VII VII VIII VIII LIST OF FIGURES Pare Steer CS—126, Total Soluble Nitrogen Content of Rumen Fluid 31 Steer 3-707, Total Soluble Nitrogen Content of Rumen Fluid 62 Steer CS-lES, Trichloroacetic Acid Nonprotein Nitrogen Content of Rumen Fluid Steer 03-136, Trichloroacetic Acid Nonprotein Nitrogen Content of Rumen Fluid Steer 0—707, Trichloroacetic Acid Nonprotein Nitrogen Content of Rumen Fluid Steer 0-707, Trichloroacetic Acid Nonprotein Nitrogen Content of Rumen Fluid . . . . Steer CS-lio, Trichloroacetic Acid Protein Nitrogen Content of Rumen Fluid Steer 08-186, Trichloroacetic Acid Protein Nitrogen Content of Rumen Fluid Steer C-707, Trichloroacetic Acid Protein Nitrogen Content of Rumen Fluid . . . Steer C—707, Trichloroacetic Acid Protein Nitrogen Content of Rumen Fluid Steer CS-lBG, Non-heat Coagulahle Nitrogen Content of Rumen Fluid Steer CS-laS, Non-heat Coagulahle Nitrogen Content of Rumen Fluid Steer 3-707, Non—heat Cvagulahle Nitrogen Content of Rumen Fluid Steer C-707, Non-heat Coagulahle Nitrogen Content of Rumen Fluid V 54 55 66 67 Flflure XI XI XII XII XIII XIII XIV XIV XV XV C XVI A lfi Steer 03-195, Heat Coagulahle Nitrogen Content of Rumen Fluid Steer CS-126, Heat Coagulahle Nitrogen Content of Rumen Fluid Steer C—707, Heat Coagulahle Nitrogen Content of Rumen Fluid Steer C-707, Heat Coagulahle Nitrogen Content of Rumen Fluid Steer CS-l26, Alcohol Nonprotein Nitrogen Content of Rumen Fluid Steer CS-126, Alcohol Nonprotein Nitrogen Content of Rumen Fluid Steer C-707, Alcohol Nonprotein Nitrogen ontent of Rumen Fluid Steer C-707, Alcohol Nonprotein Nitrogen Content of Rumen Fluid . Steer CS-126, Alcohol Protein Nitrogen Content of Rumen Fluid . Steer CS~126, Alcohol Protein Nitrogen Content of Rumen Fluid . Steer 0-707, Alcohol Protein Nitrogen Content of Rumen Fluid . Steer C-707, Alcohol Protein Nitrogen Content of Rumen Fluid Steer CS-126, Rumen Fluid Flow Time Steer CS- 26, Foam Stability of Rumen Fluid Steer CS-126, Foam Height of Rumen Fluid Steer C-707, Rumen Fluid Flow Time vi Face 68 68 69 69 59 70 7O 7O 71 71 71 72 72 73 Floure "J 4 XVI F C'teer C-707, Foam Stability of Rumen 1:11u1C1 O O I O O I O O O l O I O O O O O O 73. >< < #4 co Steer C-707, Foam Height of Rumen Fluid . . . . . . . . . . . . . . . . . . ‘1 LU vii LIST OF TABLES Table Page I Mean Values, Standard Error, and Number of Samples for the Chemical and Physical Measurements of Rumen Fluid . . . . . . . 74 II Levels of Significance Using the "t" Test 0 O 0 C O O O O O O O O O O O O O O O 76 viii _ - 21—» wwEfiJ=fig _'_"-‘ ‘—’ --‘ . 4' INTRODUCTION The anticipation of bloat has influenced many livestock men to utilize grass pastures rather than the more productive legume pastures.. This practice has resulted in economic losses estimated to be twenty million dollars in the North Central states alone. The feeding of various bloat "preventives," such as hay and chemical compounds, have resulted in additional eXpense. The many bloat studies undertaken have yielded consider- able information without delineating the physiological and/or chemical cause(s) of the malady. It is generally agreed that: (1) the incidence of bloat is increasing, (2) legumes are the worst offenders, (3) new seedings cause less trouble than the same pastures on the second and subsequent years, (A) legumes cause the most trouble during the periods of lush, rapid growth, (5) bloat occurs only rarely on grasses, (6) the in- cidence of bloat varies in different years, (7) there is a seasonal variation in the occurrence of bloat, (8) bloat is more likely to occur in certain areas than in others, (9) fatal bloat occurs in the feed lot on grain and hay as well as on pasture, (10) bloat cannot be produced experimentally at will, and (11) some animals have a greater tendency to bloat than others. h.- ..T '._ ...‘-'. First, , Second1_ from ex Thirdly be dete Whether the cau' bloat, T 8hip of 11guor h.) Several basic problems still remain to be solved. First, the specific cause(s) of bloat need to be determined. Secondly, it should be resolved whether fatal bloat results from excessive pressure, toxic materials, or other causes. Thirdly, the difference between frothy and free-gas bloat must be determined, and fourthly, bloat preventives must be developed. Whether the studies should be aimed primarily at determining the cause of bloat and secondly, finding preventives for bloat, or vice versa, remains controversial. The purpose of this study is to investigate the relation- ship of some of the chemical and physical properties of rumen liquor to the incidence of frothy bloat. REVIEW OF LITERATURE Introduction The complex problem of bloat has been a challenge to stockmen for at least nineteen centuries (Dougherty, 1953). Numerous theories of the etiology of this physiological mal- function have been postulated during this long period of time. Proof of this is demonstrated by the following factors which have been reported to influence bloating in ruminants: toxic gases, physical deficiencies, ammonium carbamate, chemical constituents of plant origin, histamines, antigens, anatomical defects, rumen flora changes, excessive gas formation, min- erals, saliva, and ingesta buoyancy. Physiology of Bloat Bloat, also referred to as hoven or tympanites, is an overdistention of the rumen with gas (Dukes, 1949). It is obvious that this disorder results from.the inability of an animal to rid itself of the gas produced through rumenal fermentation. Many studies have been undertaken to determine the cause of this eructation failure. Weiss (1953b) stated that an ordinary rumen contraction, which is essential for the mixing of ingesta, involves a rumen wave which travels backward. An eructation contraction, however, involves a wave which travels forward. Clark and Weiss (1952a) demon- 4 strated that eructation consists essentially of the movement of free gas from the dorsal rumen forward and downward to the cardia. This is accomplished by: (1) a wave of contraction that starts at the posterior blind sac and moves forward, (2) clearing of the cardia, which is covered by ingesta in a normally full rumen, by a sudden relaxation of the reticulum, and (3) opening of the cardia and passage of the gas up the esophagus. Dougherty and Meredith (1955) recently completed cine- fluorographic studies of the rumen and reticulum and of eructation. They report that the following mechanisms are important in eructation: (1) the clearing of ingesta from the reticulum by two contractions of this organ, (2) contrac- tion of the ruminoreticular fold up to (or nearly to) the level of the cardia, holding the rumen ingesta away from the cardia and preventing it from flowing or falling back into the reticulum, (3) relaxation of the relatively empty reticulum, (h) contraction of the rumen, pushing gas forward around the cardia and down into the relaxed reticulum, and (5) synchronous relaxation of the cardia and diaphragmatic sphincter, permitting gas to rush into the relaxed esOphagus. It is obvious, therefore, that a blocking of any one or more of these phases could result in eructation failure and resulting bloat. Weiss (1952) stated that the main stimulus for eructation is gas pressure in the posterior dorsal sac of the rumen. 5 He further stated that the efficiency of the reflex may depend on hereditary factors. Some of the factors affecting the reflex are: obstruction of the esophagus, frothing of the ingesta, the degree of filling of the rumen, and the posture of the animal. In regard to obstruction of the esophagus, he believeclthat the cardia is kept closed by pressure from surrounding organs since there is no evidence of a cardiac sphincter. He also found that animals with a nonfunctional reticulum were particularly susceptible to overfilling. He noted that when two liters of water were added to the rumen, eructation was prevented. This worker also found that alkalosis, as determined by the carbon dioxide combining power of the blood, could cause reticular paralysis and consequent impaired eructation efficiency. Weiss also showed that section of the right ventral branch of the vagus caused abomasal and intestinal distention with accompanying chronic frothy bloat, again due mainly to inhibition of reticular activity. Section of the left dorsal branch of the vagus resulted in only temporary impaired eructation efficiency. It was found that hydrocyanic acid, atropine, histamine, and adrenalin would decrease or inhibit the reflex. Carbamyl- choline and veratrine both caused spasm of the rumen and reticulum with consequent interference of eructation. In another study, Clark and Weiss (1952a) reported that the secretion of adrenalin due to a psychic disturbance caused mass bloating in cattle. Weiss (1953b) noted that the ratio of backward to forward contractions in the rumen changed from 2:1 to 1:1 by elevating the hindquarters of a sheep. The rumen had been first paralyzed by sodium carbonate and then stimulated to eructation by introduction of air. He believed this posture effect to be further support for the theory that receptors in the posterior blind sac (origin of the forward wave of contraction) are more sensitive to pressure. He noted that increasing the rumenal pressure increased eructation fre- quency, and that complete inability to eructate was remedied by removing three liters of ingesta. As soon as the ingesta were restored, the animal returned to inefficient eructation. Types of Bloat It is common practice to refer to bloat as being either the free-gas or frothy type. Unfortunately, it is usually impossible to distinguish between these two types in non— fistulated animals. Consequently, the recent classification by COle.§E.El_(1955) is preferred. This classification outlines three types or degrees of bloat; namely, chronic, subacute, and acute. Chronic bloat is produced in an animal regardless of the nature or quality of the diet. It is there- fore obvious that an anatomical abnormality, such as an enlarged thymus acting to constrict the esophagus, or a ruminant suffering from peritonitis, could result in chronic bloat. Subacute bloat is distention of the rumen resulting from, and depending upon, a Specific diet. The bloated animal must return to a non-bloated state without treatment to be placed in this category. Bloat from legumes and the frothy type of feed lot bloat are examples of subacute bloat. The acute category of rumen distention varies from the subacute only in degree, acute bloat being manifested by more severe distention, frequent urination and defecation, and labored breathing. Factors Influencing Bloat Physical deficiency. Cole, Mead, and Kleiber (1942) and Cole and Mead (1943) reported that the cause of bloat is the lack of sufficient irritating material in the rumen to stimulate belching. Distention in the chronic bloater is explained by the animal having a higher threshold of irrita- bility. These workers also stated that bloat on green alfalfa or grain alone occurs because the feed cannot be packed away in the posterior rumen; the cardiac orifice becomes submerged and belching is prevented. Cole_gtm§lfl(1943b) stated that bloat from immature alfalfa pasture was most severe when the animals had been deprived of hay for #8 hours. When alfalfa hay was fed it was not completely effective but did reduce the incidence and severity of bloat. The feeding of Sudan grass hay protected the cows completely. The authors believed that the scabrous leaves of Sudan grass hay induced rumination and belching and thus prevented bloat. They stated, ”In our opinion, bloat on legume pasture is largely due to a lack of sufficient irritating roughage in the rumen." It is difficult to understand why cows do not bloat on immature succulent grasses if this theory is valid. Fur- ther argument against this theory is supported by the work of Newbold (1954) who found that all parts of the red clover plant produced bloat and stated, "It is hard to reconcile the results of the feeding tests reported here with any theory that regards bloat as being due to a lack of coarse material in the feed." Johns (1954) also found that bloat was produced at all stages of wilting of red clover and up to hay of 72% dry matter content. Thus the physical deficiency theory does not appear to satisfy the still unknown etiology of free-gas bloat. Excessive gas. Cole et a1 (1943) reviewed the earlier —.-_.I-—- works supporting this explanation for the cause of bloat. They emphasized that this theory is supported by more opinions than facts. Bloat is rarely produced from grasses; therefore, if the above theory is valid, one would expect more gas to be produced from legumes than from grasses. Espe and Cannon (1940) pointed out that there was little difference in the rate of gas formation between finely cut fresh alfalfa and bluegrass when the forages were placed in rumen fluid. They also pointed out that frosted alfalfa or bluegrass, an environ- tnental condition sometimes acclaimed to increase the fre- Quency of bloat, did not materially change the rate of gas (normation. Jacobson etmal (1942) also reported that no more 9 gas was produced in vitro from alfalfa than from bluegrass. - Cole and Kleiber (1948b) noted that the gas production from alfalfa and Sudan grass, when fed ad libitum, was about equal, but that the cows ate about three times as much of the latter. They emphasized that Cows bloat on alfalfa but not on an amount of Sudan grass which would yield as much gas as the alfalfa. Since animals rarely bloat when on a diet of hay, one would also expect, if the theory were valid, that more gas would be formed on green forage than on dry. Cole et_al (1942) stated that bloating on green alfalfa cannot well be attributed simply to excessive gas formation since more gas was produced from alfalfa hay than from green alfalfa. Additional studies of gas formation relating to the type and amount of feed ingested have been accomplished. Mead et a1 (1944) reported that the rate of gas formation varies directly with the amount of feed consumed. Cole and Kleiber (1948a) reported from a study of the rate of gas formation in the rumen that the feeding of glucose caused a more rapid increase in gas formation than starch, but the total gas produced was about the same. The rate of gas production in the rumen also appears to depend upon the activity of the rumen. Stone (1949) :beported that the ability of the ingesta from inactive rumens 'to produce gas is less than that of ingesta from active rumens. Several workers have studied excessive pressures in 10 the rumen. Weiss (1953b) reported that animals with excessive rumenal pressure still exhibited powerful eructation contrac- tions. However, abnormal pressure in the rumen during bloat must be defined. Cole and Kleiber (1945) stated that animals with rumen pressure of 60 mm. Hg were in serious condition. They used a tympanometer devised by Kleiber to measure pressure. Olson (1942) found that animals which had died of bloat had rumen gas pressures of 60 to 70 mm. Hg. Animals were insuf- flated experimentally to 100 mm. Hg with no ill effects. In a later work (Olson, 1944) dealing with both cattle and sheep, similar results were obtained. Barrentine et_a1_(l954) reported that bloated steers yielded tympanometer readings of 20 to 60 mm. Hg. Acutelv bloated steers had intrarumenal pressures of 80 mm. Hg. The results of these studies indicate that a mildly bloated animal should exhibit an intrarumenal pressure of approximately 40 mm. Hg. Dougherty (1940), as previously stated, pointed out that intrarumenal pressures in cases of acute bloat did not seem to be as great as in induced bloat, but yet the cattle died suddenly. In a later work (Dougherty, 1941), he insufflated the rumen of a sheep to a pressure of 140 mm. Hg and maintained the pressure for two minutes. In another study (Dougherty, 1942a), he pointed out that intrarumenal pressures on dead animals varied from 72 to 75 mm. Hg, thus 50% of the insufflated pressure. Dougherty (1941) noted that it took much less air to increase the intrarumenal pressure when air was forced up 11 through the ingesta than when air was introduced into the top part of the rumino-reticular cavity. Eructation was also more difficult when the former method was used. Weiss (1953a) reported that when air was introduced into the watery ingesta of sheep it was eructated efficiently, but eructation was interfered with when the free air was introduced into viscid ingesta. Clark (1950b) found it very difficult to cause retention of carbon dioxide in the rumen of sheep when the gas was introduced from a carbon dioxide cylinder. This is understandable when one considers the finding of Weiss (1953b) that animals suffering from excessive rumenal pressure still exhibit powerful eructation contractions. Cole et a1 (1942) reported that increased pressure, in itself, does not force gas from the rumen through the eso— phagus. Nichols (1951) indicated that insufflation of the rumen with gas increases belching in sheep. Dougherty (1942b) reported that intrarumenal insufflations of oxygen increased the activity of the experimentally paralyzed or non-paralyzed rumen of a cow. Recent work-by Dougherty et;al_(1955) dealing with in- sufflation of the ruminant stomach showed that there were marked individual differences in tolerance to the same gas insufflated at the same pressure. It was also noted that .increased intrarumenal pressure caused sharp rises in arterial (carotid) and venous (Jugular) blood pressures. The arterial firessure increased during insufflation and dropped again when tfiue intrarumenal pressure was released. It was further noted 12 that insufflation had a pronounced effect on blood gases. The changes were rapid and were influenced by the immediate intrarumenal pressures. It was observed that even during a short insufflation period the blood gases varied during and after eructation. The rapidity with which bloat symptoms appear and disappear on pasture can be correlated well with this information. Thus the theory of excessive gas formation and excessive pressure in the rumen does not appear to be supported by factual data. Conversely, the above mentioned studies indicate that living ruminants can withstand much higher pressures than are found in animals which have died of bloat. It also appears likely that ruminants have no difficulty eructating the gas produced in the rumen when the eructation mechanism is functioning properly. Toxic gas. Daugherty (1942a) analyzed the gas and ingesta of three feed lot steers that had died of frothy bloat and found that all gas and ingesta samples had relatively high concentrations of hydrogen sulfide. The steers had received a mixture of first and second cutting alfalfa the first week in the feed lot. Grain feeding was commenced the second week and at full feed the steers were getting 7 lb. per day of a mixture of 50% ground rye and 50% ground barley, plus supplement added at the rate of 1 1b. to 8 1b. of grain. (Dlson (1942) stated that he believed hydrogen sulfide played .a significant role in the cause and death from bloat. Ikougherty (1941) noted that the type of feed being ingested 13 had a marked influence on the amount of hydrogen sulfide present. When animals were fed good quality legume hay, the hydrogen sulfide content of the rumen ingesta was greatly increased. In a later work, Dougherty (1942a) stated that the hydrogen sulfide content of ingesta could be increased markedly by feeding proteinQrich feeds, whereas Cole gt_gl (1942) reported that the type of feed eaten did not alter the composition of rumen gas. Olson (1940c) reported that no striking differences were found between the rumenal gases produced from legume and non-legume pastures. In a later work, Olson (1944) referred to the formation of toxic gases from a high protein diet. Dougherty (1941) also noted that when a cow was fed poor quality hay, and when the rumen contents were dry, the animal could tolerate comparatively large amounts of hydrogen sulfide. When the animal had been fed good quality legume hay or freshly cut ladino clover or alfalfa, and the rumen contents were moist, the hydrogen sulfide tolerance was low. The tolerance was very low when the intrarumenal pressure was increased with air immediately after hydrogen sulfide insufflation. Dougherty also pointed out that the animal recovered much more rapidly from hydrogen sulfide intoxication than from carbon monoxide intoxication. He reported that the frothy ingesta of a heifer which died of bloat had a hydrogen sulfide content much greater than the level considered to be toxic to one experimental cow under average conditions. The total blood sulfur was approximately four times the amount normally found in blood. The animal ~1. tm’r‘r _ 5.; _ «:4:-v 14 had been on the same irrigated ladino clover pasture for six weeks prior to the day it died. Olson (1944) reported that hydrogen sulfide has been shown to be greatly increased in bloated animals. In attempt- ing to explain the origin and relation of hydrogen sulfide to bloat, he stated that the leaves and finer stems of legumes are relatively higher in sulfur than the stems of the same plants, with the leaves of alfalfa having twice as much sulfur as the stems. He also reported that more hydrogen sulfide was produced when cattle were stable fed fresh green alfalfa than when grazing. The possible connection of rainfall to bloat was pointed out. He reported on a milking herd which was pastured for seven years of low rainfall with no bloat. However, much bloating occurred during a period of normal rainfall. Olson reasoned that the plants contained more sulfur when they grew rapidly during periods of heavier rainfall. He believed that cows will bloat on plants grown in the soils containing the most sulfur. Kleiber 33mg} (1943) demonstrated that the relatively high hydrogen sulfide concentration in the rumen gas of cows on alfalfa pasture does not necessarily lead to bloat. They stated, "Most likely, therefore, hydrogen sulfide plays no very significant role as a condition producing bloat. Con- ceivably, on the other hand, this poisonous gas is involved in the fatal consequences of bloat." Cole et a1 (1945) reviewed the toxic gas theory and pointed out that the theory lacked substantiation since 15 hydrogen sulfide had not been shown to be in greater amounts in living bloated animals than in normal animals. They also pointed out that the evidence was to the contrary. However, they concluded with, "Considering the very appreciable amounts of hydrogen sulfide present in the rumen at all times, and the toxic effects when it is artificially introduced into the rumen, hydrogen sulfide may well be a contributing factor in death of bloated animals." Dougherty (1940) studied the effect of other gases upon the rumen. He stated that carbon dioxide was absorbed from the rumen and produced a marked effect upon the respiratory apparatus with relatively high pressures causing extreme dyspnea. He noted that an increase in intrarumenal pressure caused an increase in the rate of absorption of both carbon dioxide and carbon monoxide from the rumen. Dougherty noted that when carbon monoxide was present in the rumen, even in very low concentrations, and when the intrarumenal pressure was increased moderately with other gases common to the rumen, marked preprostration symptoms were produced. It was also reported that carbon monoxide was found in appreciable amounts in two experimental cows fed freshly cut ladino clover, and that carbon monoxide was the only gas of those used (carbon monoxide, carbon dioxide, methane, and hydrogen) that seemed to inhibit belching. Dougherty noted the significant fact that intra- rumenal pressures in cases of acute bloat did not seem to be as great as in induced bloat, yet animals died suddenly. 16 Thus the relevance and significance of toxic gases in the etiology of bloat remains obscure. Flavones and unknown_fgrage extracts. Ferguson (1948a) reported a factor in pastures that inhibited the movement of the rabbit gut. He noted that the factor, containing no nitrogen, was found mainly in legumes (white clover and al- falfa). Orchard grass and some other grasses contained smaller amounts. He thought that grasses contain both inhi- biting and stimulating factors with the latter usually more active. He further reported (Ferguson, 1948b) that prelimi- nary tests indicated that the addition of very small quantities of adrenalin to muscle affected by clover Juice caused recovery after a brief period. Weiss (1953b) later reported that the administration of adrenalin to sheep produced variable .results in sheep with some interference of the eructation mechanism. Johns (1954) reported that adrenalin was "defin- itely dangerous" when administered as a treatment for frothy bloat produced by stall feeding of fresh red clover. Ferguson_gt_al(l949) found a muscle-inhibiting com- pound in alfalfa to be a flavone. They noted that there was much less of the material in the plant in the third cutting than in the first. They later identified the flavone as a tricin (Ferguson g§*al, 1950). They believed additional flavones exist in alfalfa because other flavone preparations possess greater activity on smooth-muscle movements than the purified isolated flavone. «0.:- ii"- “on 9-01.? l7 Parsons and co-workers (1952), as mentioned previously, reported that death resulted in sheep 9 to 30 minutes after dosing them with 16 oz. and higher doses of birdsfoot trefoil extract. Death was prevented by administration of sodium thiosulfate. Sub-lethal and lethal doses of birdsfoot trefoil and excessive doses of Kentucky 31 fescue extracts did not produce a bloated condition. Sixteen ounces of alfalfa or ladino clover extract produced slight bloat, while 32 oz. of either extract produced excessive bloat in less than one hour. Larger doses produced scours in the experimental animal. Securing was also observed when beef cattle bloated on alfalfa- ladino pasture in Michigan (Smith and Emery, 1955b). Parsons et.al.(1953) reported that rumen contents from cows that died due to bloat inhibited the motility of the isolated rabbit intestine. The effect was also noted with forage extracts taken from the pastures that these animals were grazing at the time of death. This inhibition was not observed when the rumen contents from normal cattle were used. Acute bloat and death were produced in sheep using an extract of ladino clover. Orally administered legume extracts were noted to inhibit the eructation mechanism. Lindahl et_ a} (1954) reported that no distention of the rumen was produced when sheep grazing on grass pasture were drenched with ladino clover Juice or alfalfa Juice. Newbold (1954) reported that red clover was fractionated and 16 to 18 l.of the fractions were given by drench. Only feeds containing the hydraulically pressed Juices produced bloat. Parsons and co-workers (1955) 18 reported that an extract of ladino clover, known to cause bloat, inhibited eructation in a fistulated cow. The extract, as in the previously mentioned work by Parsons §t_a} (1953), inhibited the motility of segments of the isolated rabbit gut. The active substance was stable to heat and freezing and the material in one sample was dialyzable. Blake §t_a1 (1955) reported that acute bloat in cattle was induced by oral administration of Juices eXpelled from freshly cut al- falfa. Mild bloat was induced with concentrated and with spray dried alfalfa Juice. It is evident that in the maJority of studies concerned with bloat produced by forage extracts, the presence or absence of frothiness is unknown. A universal agreement on this factor alone would greatly aid the solution of this particular phase of the etiology of bloat. SaponinEL- Quin (1943) studied the pathogenesis of acute bloat in South Africa and found that foam was produced in the rumen of sheep fed freshly cut alfalfa. He found that pressed alfalfa and the watery extract of alfalfa, on shaking, resulted in a foam which was stable for twelve hours. The material responsible for the foaming was found to be a saponin. He pointed out that cases of rapidly fatal bloat in sheep were associated with greedy feeding of the tops of alfalfa. It was not reported from what part of the alfalfa the saponin was isolated. It was noted, however, that the sugar content of alfalfa during an outbreak of bloat increased from 2.5% in early morning to 6% in late afternoon, when the most bloat 1 w: m 5‘11 “.mv‘km 19 occurred. Evans and Evans (1949) reported that saponin in a 1:5000 dilution paralyzed rabbit intestine. They also showed that one liter of clover Juice, from about 5 lb. of fresh clover, introduced directly into the rumen of a sheep, paralyzed rumen movement immediately. They tended to incri- minate hydrocyanic acid, however, rather than a saponin as the paralyzing factor. Henrici (1952) studied legumes and grasses grown on different soils in South Africa. This worker isolated two saponins from Tribulus terrestris but found none in plants grown on soil where no bloat had occurred. Henrici believed the saponins are formed on certain soils, presumably those which are unable to supply zinc in sufficient quantity to Tribulus terrestris during periods of flush growth. This investigator reported that zinc deficiency and wilting are factors dissolving starch and leading to the accumulation of sugars. Alfalfa was also studied and yielded results similar to that of Tribulus terrestris. The saponin isolated from alfalfa foamed readily and was hemolytic, as were those from Tribulus terrestris. The saponin was not the same as Jacobson's, which was not hemolytic and contained nitrogen. Henrici reported that none of the alfalfa grown on non-bloat producing soils under similar conditions was ever hemolytic. It was pointed out that the saponin may increase the permea- bility of the intestines so as to allow sugars and gases to pass with great velocity, or the saponin may be split up in the "stomach" and a large amount of sugars freed, thus 20 increasing the amount of rapidly fermentable sugars. Lindahl and others (195%) investigated the role of alfalfa saponin in ruminant bloat. They found that large doses of saponin, 15 gm. with sheep and 75 gm. with a heifer, produced light bloat. These amounts of saponins are believed to exceed the amounts that could be ingested from alfalfa by normal grazing. They noted that distention appeared to be due to gas retention rather than froth. No distention was produced when the water allowance was increased two or three times. Potter and Kummerow (1954) studied the biological activity of saponins isolated from alfalfa and soybeans. They reported that both the purified alfalfa and soybean saponins inhibited the growth of chicks, while their genius did not. They believe that bloat in cattle from untoasted solvent extracted soybean oil meal might be explained by toxic soyasapogenols, which on cooking hydrolyze to non- toxic genins, but may not do so if the cooking process is too mild. Jacobson and Lindahl (1955) studied the rumen ingesta volume increase in frothy bloat. They found a greater increase with saponin plus glucose than with either one alone. Colvin et_al(l955) studied the effect of alfalfa saponin on rumen activity in sheep. They reported that when sheep were on diets of either Sudan grass or alfalfa tops, the administration of 100 gm. of alfalfa saponin dissolved in one liter of 5% glucose solution caused death within 1 1/2 to 2 1/2 hours. The administration of 100 gm. of saponin dissolved in one liter of 0.9% sodium chloride solution produced death 21 in 2 l/# hours in a sheep fasted 48 hours. After the admin- istration of the saponin solution, rumen motility ceased almost immediately. Frothy bloat was observed when the glucose and saponin were introduced into the rumen together. Postmortem findings indicated that death was associated with an intense submucosal and subserosal hemorrhagic conditiOn of the gastro- intestinal tract. In other trials, rumen motility was mark- edly depressed by either 25 gm. of alfalfa tops or 12.5 gm. of alfalfa saponin. They noted that when alfalfa tops were fed, the animals became slightly bloated; however, no disten- tion was observed following the use of alfalfa saponin in the animals on an oat hay diet. The preceding review indicates the possible relationship of saponins to the cause of ruminant bloat. It is obvious, however, that cattle and sheep probably do not ingest as much saponin under normal grazing conditions as was adminis- tered in the bloat-producing experiments. It is well to keep in mind, however, that the hemolytic effect of alfalfa saponins disappeared very rapidly after the compounds were extracted (Henrici, 1952). _§ydrocyanic acid. Weiss (1952) stated that small doses of hydrocyanic acid inhibited the reticulum and backward movement of the rumen, with consequent inefficiency of eruc- tation and abolition of the eructation reflex. Clark and weiss (1952a) noted that sub-clinical doses may cause bloat whereas large doses cause total rumenal paralysis. 22 Clark and Quin (1945) showed that more than four times the amount of potassium cyanide is required to cause rumenal paralysis in sheep during active fermentation of alfalfa in the rumen than after a fast of fourteen hours. The increased tolerance of potsssium cyanide after feeding was explained on the basis of an accelerated elimination of hydrogen cyanide from the lungs. This results from the greater respiratory exchange, which in turn is caused by the absorption of carbon dioxide from the alimentary tract during fermentation. They reported that sheep showing paralysis of the rumen, caused by potassium cyanide, were able to eructate two liters of gas per minute introduced through the rumen fistula. They con- cluded with, "These observations, therefore, afford no evidence for incriminating the cyanogenetic factors in plants as being associated with the etiology of acute bloat in ruminants." ~Later work by Clark (1951) showed that rumen musculature paralyzed by potassium cyanide failed to respond to carbamyl- choline-chloride. The drug combined its effect with prussic acid to increase the severity of the symptoms of prussic acid poisoning. Evans and Evans (1949) showed that one liter of clover Juice, from about five pounds of fresh clover, introduced directly into the rumen of a sheep paralyzed its movements immediately. They noted that the condition resembled hydro- cyanic acid poisoning. They then starved a bull and two heifers for twelve hours and turned them onto white clover pasture. The animals quit grazing the clover after twenty 23 minutes and grazed rough herbage other than clover. The bull bloated but the heifers exhibited only mild distention. The bull had 0.1 mgm.% hydrocyanic acid in its blood; the heifers had 0.0# mgm.%. Heath and Park (1953) studied an irreversible choline- esterase inhibitor in white clover. They stated, "Clover may in some cases produce signs of anti-choline-esterase poisoning as well as cyanide poisoning. We think, therefore, that these observations may have some relevance to bloat." Parsons e§_al (1952) reported that dosing sheep with 16 oz. of birdsfoot trefoil extract produced death in 9 to 30 minutes. They noted that the symptoms suggested hydrocyanic acid poi- soning. Dougherty and Christensen (1953) found that freshly pressed birdsfoot trefoil Juice contained at least twenty times as much hydrocyanic acid as freshly pressed alfalfa, ladino clover, or orchard grass. One and one-half liters of each of the Juices were introduced into the rumen of sheep. Only the birdsfoot trefoil extract proved lethal, and no symptoms of bloat were noted. The lethal dose of birdsfoot trefoil, for sheep, was found to be between 212 and 318 milliliters. The introduction of 350 ml. of cyanide-free birdsfoot trefoil extract into the rumen of a sheep did not prove lethal. Dougherty and Christensen pointed out that even though hydrocyanic acid is toxic to sheep, there is little or no clinical evidence that either hydrocyanic acid poisoning or bloat occurs in animals pastured on birdsfoot trefoil. 2A Ammonium carbamate. Hale and King (1955) cited the bloating of sheep, observed by Repp_gtflal (1955), when urea was administered orally in doses that were fatal or near fatal. They believed that conditions in the rumen at this time were similar to those when ruminants suffer from acute bloat on immature legume pastures, since these pastures are high in nonprotein nitrogen. They stated, "In consideration of results in this experiment, it appears plausible that death during acute bloat in cattle and sheep may actually be due to the absorption of ammonium carbamate or some related nitrogen compound." Clark_etnal (1951) showed that in acute cases of urea toxicity in sheep, clinical symptoms appeared from 30 to 60 minutes after dosing. Dullness was followed by marked hyper- esthesia and severe muscular twitches over the whole body. Moderate to severe'ukfiting frequently occurred at this stage. The introduction of urea into the rumen was followed by a decrease or entire cessation of rumenal motility and a sharp rise in the pH. It is significant that sheep on a diet of poor quality grass hay were more susceptible than those on alfalfa hay, thus tending to contradict the ammonium car- bamate theory. However, the cause of rumenal paralysis was associated with an increased alkalinity of the rumenal contents following the formation of ammonia. The paralysis could be prevented or alleviated by administration of an acid. The toxicity of urea was found to depend on the activity of the rumenal flora, as determined by the basic diet, and the presence 25 of available carbohydrate. The validity of the ammonium carbamate theory appears to depend upon the speed with which ammonium carbamate in the blood is hydrolyzed to ammonium carbonate by carbonic an- hydrase. Sumner and Somers (1943) stated that carbamate slowly hydrolyzes to carbonate and that hydrolysis is retarded by carbon dioxide. They believed that the first products formed from urea by urease, known to be present in the gastric mucosa of cows and sheep, are carbon dioxide and ammonia. In the ab- sence of buffers, these unite to form ammonium carbamate. If buffers are present, the products are ammonium salts and carbonic acid. It is noteworthy that carbonic anhydrase is inhibited in the dark by carbon monoxide and hydrogen cyanide (Sumner and Somers, l9h3). The latter compound has been incriminated as contributing to the cause of bloat (Clark and Quin, 1945; Evans and Evans, 1949; Clark and Weiss, 1952a; and Weiss, 1952). Histamines and allergies. Dougherty (l9h2a) reported that all ingesuisamples were relatively high in histamine content, after studying steers that had died of frothy bloat while on feed lot. He reasoned that the histamine may have come from decarboxylation of histidine. Dougherty cited the fact that the histamine content of rumen ingesta can be increased markedly by feeding protein-rich feeds. He also reported (1942b) that histamine and adrenalin had a marked , fl—ew an.— 225 depressing action on rumenal motility. Petersen (1945) found that the intravenous inJection of histamine produced marked bloat. Clark's study (1950a) of the action of histamine on rumenal musculature showed that the intravenous inJection of histamine (1 to 2 ngb.) aaused a prompt and complete cessation of rumenal movements without affecting the general intrarumenal pressure. The large intestine, however, dis- played hypermotility. The inJection of three antihistaminic drugs reversed the.effect of histamine, and prior adminis—' tration of the drugs prevented histamine action, to prove the above effects were due to that specific compound. Two milli- grams of histamine were intravenoushfinJected after left vagotomy. The rumen and reticulum gave no response, but the intestine assumed a wrinkled appearance, due to spasm of the circular muscle layer. The distal end of the left vagus was then stimulated with the previously determined strength of current, and the reticulum and rumen were seen to contract as strongly as they had prior to the inJection of histamine. Repeated stimulation at intervals of from 2 to 20 minutes yielded identical results. Contraction was not observed in either the rumen or isolated strips of the rumen. Clark stated, ”Rumenal stasis associated with high protein intake may, therefore, either be due to excess alkalinity of the rumen (ammonia) or to the presence of toxic amines (hista- mines ?), or to a combination of both these factors." Weiss (1953b) PePorted the blocking of eructation in sheep by the iliJection of histamine. 27 Dougherty and Cello (1952) reported a study of toxic factors in the rumen ingesta of experimentally overfed sheep. Coarsely Cracked grain, placed in the rumen, caused 5 of 6 sheep to die in 20 to 24 hours. Heparinized blood plasma taken from acutely sick sheep depressed the blood pressure of an- esthetized dogs. Freshly drawn normal heparinized sheep plasma did not. InJections of the toxic rumen ingesta mat- erial caused a pronounced leukopenia in dogs. The leukopenia coincided with the fall in blood pressure, but occurred in a refractory dog when the blood pressure fall did not occur after an inJection of ingesta fluid. There was an indication that the toxic factor was absorbed from both the intestinal tract and the rumen. Three antihistamines gave fair pro- tection. Johns (1954) reported that an antihistaminic was "definitely dangerous" when used to treat frothy bloat produced by feeding red clover. In an earlier work, Dougherty and Cello (1949) reported that the toxic factor depressed blood pressure in dogs and goats, inhibited rumen motility in all intact animals, stimulated motor activity of the lower gut in the intact dog and goat (anesthetized) and the sheep (unanesthetized). Clark (1950a), as previously reported, showed that histamine also stimulated motor activity of the lower gut of sheep. The toxic substance was heat stable, dialyzable and not volatile with steam distillation at varying pH levels. ' It is noteworthy that the inJection of histamine in humans causes a fall in blood pressure (Anonymous, 1952). :28 An abnormal distribution of leukocytes results, with leuko- penia occurring in the peripheral blood and leukocytosis in the blood of the abdomen (Houssay_et;al, 1951). A know- ledge of the corresponding influence of histamine on the blood of ruminants would aid the study of the etiology of bloat. The rumen distention produced by Trichomonas fetus antigen inJection has resulted in bloat. Kerr and Lamont (1946) and Lamont (1946) reported this phenomenon after the antigen was inJected into the uterus of cattle. The symptoms of bloat appeared after the second, or exciting, dose of antigen. They believed the bloat resulted from a spasm of "unstriped muscle at the esophageal opening into the rumen." The allergic shock was relieved by inJecting 3 to 5 cc. of ad- renalin subcutaneously or administering atropine sulfate in 0.5 grain doses. Shanks (1946) also reported the production of bloat after Trichompnas fetus antigen inJection. He stated that this rumen distention in cows in Northern Ireland resulted from a form of protein shock, and it was again re- lieved by the inJection of adrenalin. He noted that adren- alin inJection also "cured" cases of naturally occurring bloat. It is noteworthy that Johns (1954) found adrenalin therapy on frothy bloat to be "definitely dangerous," while Weiss (1953b) found that it interfered with eructation, and Clark and Weiss (19523) reported that adrenalin inhibited the reticulum. Shanks (1946) proposed the theory that young, 29 luscious pastures and certain foods contain specific proteins whidicause protein shock on absorption into the body. He also believed that the shock interferes with belching, possibly by contracting the sphincter at the cardiac orifice. In regard to feeding certain proteins, Dougherty (1942a) stated that the histamine content of rumen ingesta can be increased markedly by feeding protein-rich feeds. Clark (1950a),as previously cited, reported that rumenal stasis, associated with a high protein intake, may either be due to excess alkalinity of the rumen, or to the presence of toxic amines, or to a combination of both these factors. Quin and Van Der Wath (1938) reported that the type of feed had little influence on the motility of the rumen. It is obvious that the fractionation and separation of proteins must be accomplished before the protein shock theory can be substan- tiated. ' Saliva. Smith (1954) showed that frothy bloat could be induced in an animal by cannulating the parotid glands and thus preventing a portion of the serous saliva from entering the rumen. It was postulated that the mucoid type of saliva caused the ingesta to froth, whereas the serous saliva decreased froth formation. McGilliard (1955) found two distinctly different types of saliva in cattle. When collection was made at the cardiac end of the esophagus, the saliva was decidedly more viscous than when collected orally from the same cow. Komarov and Stavraky (1940) studied the submaxillary saliva of cats. They reported finding two kinds 30 of saliva, one produced by chorda tympani stimulation and the other by administration of adrenalin. The two kinds of saliva contained a different and characteristic glucoprotein. They believed that chorda tympani stimulation caused the secretion of one glucoprotein from mucous cells of the sub- maxillary gland, and that adrenalin administration caused a different glucoprotein to be secreted by the serous cells of the gland. They found that urea represented quantita- tively the main fraction of the nonprotein nitrogen of sub- maxillary saliva by either form of stimulation. They believed that prolonged chorda tympani stimulation caused a decrease in the permeability of the submaxillary gland to the passage of nonprotein nitrogen, whereas adrenalin increased the permeability. Quin and others (1951) reported a finding that may well be attributed to saliva. They noted that rumenal ingesta invariably became progressively more watery during fasting, despite the fact that the water consumption was always greatly reduced. Clark and Weiss (1952b) studied reflex salivation in sheep and goats. They noticed that in- gesta became more watery after the feeding of hay, even in the absence of drinking water. They felt that the decrease in viscosity could only be explained by increased salivation and proceeded to determine if an increase'occurred. They first located the afferent arc of the salivation reflex by stimulating the proximal and distal ends of the severed left vagus nerve. Electrical stimulation of the proximal end of the left vagus yielded profuse parotid gland secretion whereas 31 there was none when the distal stump was stimulated. Mani- pulation of the reticulum also caused an increased flow of saliva. Mechanical stimulation of the throat caused a two- fold increase in parotid secretion. Mechanical stimulation of the cardiac region produced a four to five-fold increase, provided the vagi were intact. Division of the vagi abolished this reflex and reduced the resting flow. Thus it can be seen how feeding may decrease the viscosity of the ingesta through the action of the parotid gland. Weiss (1953a) studied the significance of reflex salivation in relation to froth formation and acute bloat in ruminants. He found that frothiness was produced in sheep when the rumenal ingesta became thick and viscid; conversely, the amount of froth was insignificant when the rumenal ingesta were watery. He noticed that the feeding of crisp, succulent, prebloom alfalfa caused the rumen ingesta to be thick and viscid with bloat resulting from frothing of the thick ingesta. When mature stalky al- falfa was fed,the rumenal ingesta immediately reverted to a watery consistency, even in the absence of drinking water, and bloat ceased. Similar results were obtained by placing chopped hay in the rumen. He stated, "The conclusion is Justified that the rapid reduction in consistency of the 'rumenal ingesta after feeding stalky lucerne was due to reflex stimulation of salivary secretion initiated in the forestomachs by the physical character of the feed. Ingestion of stalky alfalfa was also slower with the result that proportionately more saliva is secreted per given weight of feed." One may 32 argue that the decrease in consistency of the ingesta could have been due to:hvxeased serous saliva secretion, and the increase in consistency due to increased mucoid saliva, rather than difference in total saliva as Weiss postulated. Weiss (1953a) also pointed out that free air was eructated effi- ciently when introduced above both viscid and watery rumen ingesta. Air introduced into watery ingesta was also eruc- tated efficiently, but eructation was interfered with when introduced into viscid ingesta. Several other reports indicate possible relationships between saliva and bloat. Cole et_al_(l945) pointed out that saliva is a potential source of rumen carbon dioxide and may transport daily to the rumen an equivalent of 120 liters of carbon dioxide. Clark (1944) pointed out that a sufficently fluid state of the rumenal contents is also essential for maintaining rumination. Jacobson and others (1942) and Espe and others (1943) attempted to answer the question of why cattle appeared to bloat more on frosted pasture and pasture covered with dew. They postulated that less saliva was added to the rumen when these types of pasture were ingested. Balch 33:11 (1953) later reported the amounts of saliva produced under slightly different conditions. They found that a cow, during eating, produced saliva at the rate of 5.8 lb. per 1b. of hay, when receiving limited amounts of water, but only 4.6 lb. of saliva per 1b. of hay, when re- ceiving water at will. This work appears to confirm the findings and beliefs of other workers who have been previously 33 mentioned (Quin etma}, 1951, Clark and Wiess, 1952b, Weiss, 1953a)- Buoyancy. This explanation for bloat is similar to anv obsolete theory reported by Jacobson et_al (1942), Espe etwal (1943), and Cole et_§l (1942). The difference lies in the belief that in the modern theory, some ingested material is buoyed on the surface of the rumen liquor, whereas other in- gested material sinks, raising the rumen liquor level and thus exerting a corresponding blocking influence on the cardiac orifice (Anonymous, 1953). In the older theory, there was simply a physical blocking of the cardiac orifice by compacted rumen ingesta. Nichols et al (1955) attempted to substantiate the modern theory by studying the effective buoyancy of the rumen Juice of cattle fed hay, grass, and fresh legumes. They postulated that if enough small bubbles are produced in the lower regions of the rusem, they may be retained in the rumen fluid with the resulting development of a frothy mass. They believed that the effective buoyancy of rumen Juice can be expressed on the basis of its specific gravity. They also believed that the frothy ingesta has less density and less buoyancy. They reasoned that the contents of the ventral sac of the rumen should be lower in specific gravity following the intake of fresh legumes than following the intake of hay or fresh grass. Their determinations showed that approximately one-half hour after the intake of feed and water, the Specific gravities of ventral sac liquor were significantly lower than 34 those of the corresponding samples taken before feed and water ingestion. Specific gravities of centrifuged samples one- half hour after feeding were altered the least when legumes were fed and water intake was lowest, and were significantly reduced when hay and grass were fed and water intake was greatest. Specific gravities of strained rumen liquor one- half hour after intake of legumes were significantly lower than after the intake of either hay or grass. This lowering was evident even in the absence of the apparent lowering effect of water intake, as observed on the specific gravities of centrifuged half-hour samples. Abnormal rumen flora. Hungate et a1 (1952) studied microbiological changes associated with acute indigestion in sheep. They found that when an excess of grain or glucose was introduced into the rumen, it caused a marked change in the rumen microorganisms. The cellulolytic bacteria were greatly decreased in number, the protozoa were killed, and the relative number of gram-positive bacteria increased. They also noted that nonvolatile acids ascumulated and the amount of volatile acids diminished. R‘unen motility was par- tially or completely inhibited. Dougherty and Cello (1949) reported an increase in volatile acids of the rumen may be associated with acute indigestion. Quin et’al_(l95l) studied the effect of fasting on the activity of the rumenal flora of sheep and cattle. They 'reported that fasting caused a marked decrease in the ability 35 of the rumenal flora to ferment glucose, and thus presumably decreased gas production. Cellulose digestion returned to a high rate rapidly, after fasting, if grass hay was fed. The recovery was slow in sheep, however, if alfalfa hay was fed. Stone (1949) reported that during fasting the volatile acidity of bovine rumen ingesta decreases, and is also subnormal in atonic rumens. Jacobson and Lindahl (1955, studied the biochemical, physical, and bacteriological factors involved in feed lot bloat. They found that when cattle were on a hay and corn silage diet, the ratio of acetic, propionic, and butyric acids was roughly 6:2:1. After the cattle were on a diet of 4 lb. of hay and 6 lb. of a bloat producing mix for four weeks, the ratio was altered to approximately 7:1:1. They reported that the bacterial content of the rumen markedly increased when the animals were on the bloat diet. They also noted a high degree of encapsulation of the rumen bac- teria which increased with the incidence of bloat. They be- lieved this encapsulation played a part in the stability of the froth. Cole et_al (1955) recently reported similar ob- servations and conclusions. Smith 22,21.(1953) reported the appearance of an iodophilic streptococci concurrent with frothing of rumenal ingesta. The frothy bloat was experi— mentally produced with a grain mix containing corn and soy- bean oil meal. Attempts to find the cause of bloat through rumen microflora studies have generally been discouraging. The 36 possible significance of increased bacterial encapsulation concurrent with frothy bloat may, however, contribute to the solution of the bloat problem. Minerals and soil factors. Henrici (1952) is convinced that a soil factor is the primary cause of the involved phenomenon of bloating. This investigator believes, speci- fically, that the cause of bloat is related to a zinc defi- ciency in bloat-producing plants at the time of lush growth. It was stated that zinc deficiency only occurs under high illumination and with high temperatures, never in a foggy country. It was also pointed out that the role of zinc in the plant is not fully understood, but that only the results of the deficiency are known. They are: stoppage of growth, resulting in dwarf plants; increase of peroxidase in the leaves; destruction of the auxin and chlorophyll, accelerated by peroxidase activity; dissolution of starch, but no decrease in sugars; and the appearance of phytosterine and pclyphenolic substances. Henrici grew Tribulus terrestris in plots where no bloat had ever been produced,with rain as the only source of water. Thus, a zinc-free condition was presumed to have existed. Analysis of the fresh plants showed that the stems contained more starch than the leaves; sugars were present in approximately the same quantity as in fresh summer legumes; but starch occurred in much larger amounts, particularly in the stems. It was noted that in fresh Tribulus terrestris, there was a tendency for monosaccharides to prevail. A saponin-like glucoside was extracted for the plants that 37 caused bloat in ruminants. The glucoside was not present in fresh plants from ordinary soil in an area where no bloat had been known to occur. Henrici stated that the saponin was formed on certain soils, presumably those which are deficient in zinc or which are unable to supply zinc in sufficient quantity to Tribulus terrestris in flush periods. It was further hypothesized that at the end of the season, no zinc deficiency occurs, as growth is slow, and no saponin is formed. The possibility of the saponin being a phytosterol was ex- pressed, and Reed and Dufrency (1942), who made mention of a phytosterin being formed when a zinc deficiency and strong illumination occur simultaneously, were cited. McIntosh (194l)‘reported that cattle could be pastured on alfalfa grown on fertilized and "properly farmed" land without encoun- tering bloat. He cited cases where cows that had been bloating on poor soils were moved to well-fertilized soils and bloat- ing ceased. Duckworth and Godden (1939) found that inorganic phos- phorus, total calcium, ionic calcium, calcium in a phOSphate- carbonate ccmplex, calcium in an unknown complex form, protein bound calcium, and total magnesium of the blood appeared in lesser amounts when the cow was in a bloated state than when the animal was apparently normal. Other workers have studied minerals and soil factors relating to bloat. Espe and Cannon (1940), in an attempt to prevent bloat, found that salt, soda, hydrated lime, or ‘5 38 combinations of the three which the cow would tolerate in her drinking water increased rather than suppressed gas for- mation. Olson (1940b) reported that bloating of cattle on alfalfa in Argentina was not considered a serious menace when rock salt was available at all times. In another work (1940a), he reported that steamed bone meal, salt, and baking soda in the drinking water of cattle had no effect on bloat production. Cattle were observed eating dirt Just prior to bloating while on alfalfa-ladino clover pasture (Smith and Emery, 1955.) Other cattle were observed eating dirt after they had been bloated by a grain mixture (Huffman, 1955). Heredityi The possibility that the tendency to bloat may be inherited has been reported by several workers. Knapp et_al.(1943) reported that a highly significant differ- ence in the number of steers showing excessive bloat was ob- served among the progeny of various bulls. Their analysis was based upon the number of steer days during which bloat occurred. Johns (1954) studied bloat on red clover with two pairs of identical twins and a single cow. He observed similarities between twins and differences between pairs in bloating behavior, reaction to different periods of star- vation, and reaction to change in dry matter content of the clover. Hancock (1954) studied bloat in relation to grazing behavior. He also stated that there was evidence of bloat being inherited. Some twins did not bloat on either controlled or "break" grazing, even though most of the animals did. 39 Hancock postulated that the incidence of bloat may have been due to differences in the rate of grazing and also to the type of herbage selected. He disproved this idea by feeding two sets of twins in stalls. The twins that were highly susceptible bloated forty-five minutes after being fed 25 1b. of preflowering alfalfa, but the non-susceptible twins did not bloat. Lindahl and Davis (1954) studied some factors in feed lot bloat. They noted a significant difference among the test animals in their susceptibility to bloat. Dougherty 312.3} (1955) studied the physiological effects of insufflation of the stomachs of sheep. They observed marked individual differences in tolerance to the same gas, insufflated at the same pressures. Weiss (1953b) studied the physiology of eructation in ruminants. He postulated that the eructation reflex is initiated by gas pressure in the rumen, and that reflex variations among individual animals is due to dif- ferences in the level of nervous reaction. Anatomical defects. Cole et_al (1945) pointed out that bloat in some cases might be due to a partial obstruction of the esophagus by enlarged mediastinal lymph glands. Ascott (1946) reported five cases of bloat resulting from blocking of the esophagus by tuberculosis and other diseases. Preventive Measures Prior feeding of hay. One of the most publicized meas- ures of bloat prevention is the feeding of hay prior to pasturing ruminants. Cole, Mead, and Regan (1943b) reported 40 the production and prevention of bloat in cattle on alfalfa pasture. They noted that bloat from immature alfalfa pasture was most severe when the animals had been deprived of hay for 48 hours. They found that feeding alfalfa hay prior to pasturing reduced the incidence and severity of bloat, but was not completely effective as a preventive. Prior feeding of Sudan grass, however, protected the cows completely. They also found that pasturing on Sudan grass at night almost completely prevented bloat in cows pastured the following day on alfalfa. Cole_et~§l (1943a) studied the prevention of bloat and found that supplemental feeding of hay in dry lot at night and in the pasture reduced the incidence of bloat. Cole and Kleiber (1945) reported that Sudan hay was only effective in prevent— ing bloat when animals had all they would eat overnight before being pastured on alfalfa. They found that 17 lb. of Sudan grass hay completely prevented bloat. They also noted that supplemental feeding of Sudan grass hay generally increased consumption of green alfalfa, if no concentrates were fed. Mead, Cole, and Regan (1944) reported that the feed- ing of barley straw prior to pasturing was not effective in preventing bloat. They noted that bloat occurred sooner after animals were turned into pasture if grain was fed Just pre- viously. Johns (1954) studied bloat in cattle on red clover and found that the feeding of hay was not effective in preventing bloat. He noted bloat was produced at all stages of wilting of clover up to hay of 72% dry matter content. Olson (1940a) also reported that prior feeding of hay was not effective in 41 preventing bloat. It is well to keep in mind that most le— gumes produce bloat and most grasses do not. Consequently, one might expect a grass hay to prevent bloat, but not a legume hay. The previously reported studies seem to bear this out. Soiling. Another measure that has been employed to prevent bloat is soiling, or cutting the feed green and hauling it to the cattle. Newbold (1954) reported that red clover pastures producing bloat with grazing would also produce bloat when out and stall fed. Johns (1954) reported that bloat was produced when red clover at all stages of wilting was stall fed. Thus it can be seen that the soiling mefllod is-not completely effective in preventing bloat. Alternate grazing of grass_and legume pastures. Cole -8EJEl_(1943b) found that overnight pasturing on Sudan grass proved effective in preventing bloat on alfalfa pasture the following day. Simultaneous pasturing of Sudan grass and a legume did not prove practical, however, as a method of preventing bloat (Regan and Mead, 1945). Intermittent versus contkuous grazing. Hancock (1954) reported more cases of bloat when the cattle were allowed to graze over several periods rather than one. The bloat, however, was mostly mild. Six out of eight days of observation, the Tweak-grazed cows suffered from more bloat than the contin- Llcusly grazed control group. Thus the break-grazing method C>f preventing bloat did not appear to be effective. 42 Proper grass-legume ratio. Cole et*al (1943a) reported .—--—- -- - ~4 that bloat rarely occurs if grasses make up 50% of the pastwre mixture. Hancock (1954) reported in his study of bloat in relation to grazing behavior that, ”We‘still need to know the proportion of grasses necessary to make a mixed clover- " and "We need to know whether there are grass sword safe, specie or strain differences among grasses and clovers in their relative bloat danger." Grazing maturegplants. A sixth measure employed to prevent bloat is prohibiting grazing until legumes reach a mature stage. Beef cattle in Michigan bloated on seed stage alfalfa and ladino clover pasture (Smith and Emery, 1955). Hancock (1954) reported less bloat on the most mature pasture, (but admitted that the critical stage of maturity was unknown. Johns (1954) reported bloat on red clover hay of 72% dry matter. Oil sprays. The spraying of pastures with a peanut oil emulsion has resulted in the prevention of bloat in New Zealand (Petersen, 1955). Two ounces of corn oil were emul- sified with 2 qt. of water and sprayed on 9 by 14 yd. plots in Michigan (Smith and Emery, 1955a). Bloat was not pre- vented, but there was an indication that froth formation was impaired. Additional studies must be completed before the effectiveness and practicability of this method can be a. scertained . Feeding antifrothing_gompounds. Barrentine, Shawver, E1nd Williams (1954) reported that the administration of 20 gm. cEipsules of methyl silicone before morning grazing helped to 43 prevent bloat in the morning but not in the afternoon. A recent study in Michigan (Smith and Emery, 1955a) showed that prior feeding of 5 gm. of methyl silicone or spraying a methyl silicone emulsion on small plots was not effective in preventing bloat. Household detergents have also been fed to cattle in attempts to prevent bloat (Anonymous, 1953). Feeding minerals. Most of the data indicate this prac— tice to be of doubtful value. Olson (1940b) reported that the bloating of cattle from alfalfa in Argentina is not considered a very serious menace when rock salt is available at all times. Espe and Cannon (1940) reported that the amounts of salt, soda, and hydrated lime, or combinations of the three, which cows will tolerate in their drinking water increase rather than suppress gas formation. Regulating water consumption. Lindahl et a1 (1954) produced bloat in ruminants by feeding large doses of sapo- nins, but no distention was produced if the amount of water was increased two to three-fold. Olson (1940a) reported that cows were watered four times in ten hours while on pasture and still bloated. Cole, Mead, and Regan (1943b) studied the production and prevention of bloat in cattle on alfalfa pasture. They stated, "Although bloat has often been attri- buted to either the presence or the absence of water, we tobserved no differences in the extent of bloat which we could iascribe to this factor. Our cows always had free access to \mater when they were taken off pasture, and very few bloated Etfter that time." Weiss (1953b) reported a study in which 2m test animals were deprived of water for twenty-four hours before pasturing. Half of the animals were watered Just prior to turning on pasture. A greater incidence of bloat among the watered group was noticed. In another study, Weiss (1953a) found that frothiness occurred in sheep, when the rumen ingesta were thick and viscid. The froth was insig- nificant, however, when the rumen ingesta were watery. If this observation is valid in all cases of bloat, then one might expect thick and viscid froth-producing ingesta to become non-frothy when the animal takes in water. It is also apparent that when ingesta are too dry to produce froth, the ingestion of water may create froth within the rumen. It is also well to keep in mind that the rumen ingesta may become more watery in the absence of drinking water (Quin_et.al, 1951, Weiss, 1953a). Correcting anatomical abnormalities. It is obvious that bloat resulting from anatomical abnormalities and faulty nervous mechanisms can best be controlled by medically cor- recting the respective abnormalities. Treatments for Bloat Turpentine or kerosene. Whether the above compounds act as irritants to stimulate belching, or alter the surface ‘tensicn of rumen liquor to allow the gas to escape, or both, :13 still controversial. Dougherty and Meridith (1954) reported Ein‘ig gifgg study of the efficacy of silicone products com- Efiared to turpentine for the treatment of bloat. They found 45 that the silicone products dispersed rumen liquor foam better than turpentine. The reverse was true, however, when the materials were Judged against the reconstitution of foam produced by reshaking the rumen liquor. Clark (1948) found that the in vitro administration of turpentine breaks up foam and facilitates the movement of gas through rumen in— gesta. Blake_gt_al (1955) reported that the in_viyg admin- istration of turpentine decreased the surface tension of frothy rumen ingesta. Clark (1950b) reported that turpentine, administered to sheep bloated by 100 gm. of sucrose and 2 gm. of saponin, resulted in a moderate rise in rumen pressure. Formaldehyde. This compound has also been successfully used to treat bloated animals (Huffman, 1955). Lipase treated cream. Smith_et a1 (1953) reported that lipase treated cream was effective in reducing the froth of experimentally produced frothy bloat. - Silicone compounds. Dougherty and Meridith (1954) found that silicone suspension products were effective in dispersing in_vitrg_rumen liquor foam preparations. They were not as efficacious as turpentine, however, in preventing reconstitution of the foam. Quin’etial (1948) reported the aiccessful treatment of bloat by intrarumenal inJection of Inethyl silicone. Smith et_al_(1953) reported the reduction of froth in experimentally produced frothy bloat by adminis- tering methyl silicone. Barrentine and Shawver (1954) re- ported that treating bloated animals with methyl silicone Eappeared to be of doubtful value for bloat produced by pasturing animals on ladino clover. Household detergents. These cleaning compounds have been used to treat and prevent bloat (Anonymous, 1953). Blake et a1 (1955) found that the in vivo administration of a.detergent decreased the intrarumenal surface tension. Adrenalin and atropine sulfate. These compounds have been used successfully to relieve specific cases of bloat produced by inJection of Trichomonas fetus antigen (Kerr and Lamont, 1946, and Lamont, 1946). Shanks (1946) also relieved the antigen type of bloat and also "naturally occurring" bloat by inJecting adrenalin. Clark and Weiss (1952a), llowever, reported that a mass outbreak of bloating in cattle :resulted from adrenalin secretion, caused by psychic dis- turbance of the animals. Johns (1954) reported that the iadministration of adrenalin to cattle bloated by stall fed red clover was "definitely dangerous." Treatment with an anti- Ihistaminic agent yielded similar results. Mechanical treatments. The administration of a stomach tube, or trocar and cannula, to a bloated animal is commonly employed (Cole ef;al, 1945). The latter treatment should be attempted only if a bloated animal is down and unable to get up. Emergency rumenotcmies sometimes have to be resorted to to :relieve severe cases of frothy bloat (0016.52451’ 1955). Mild <2ases of bloat have been relieved by simply walking the animal «or placing a stick or rope in the animal's mouth (Petersen, 1950). ‘P EXPERIMENTAL PROCEDURE Steer number C-707, a rumen fistulated, five year old, 1,450 lb. Guernsey, was placed on each of two rations two 'weeks prior to the initial sampling date. The first ration consisted of 24 lb. of first cutting alfalfa hay plus 50 gm. of salt daily. The second ration consisted of 4 lb. of second cutting alfalfa hay, 50 gm. of salt, and 10 lb. of the follow- ing concentrate mixture daily, subsequently referred to as T-7 mix: % ground corn % soybean oil meal % dicalcium phosphate % salt % calcium carbonate m./1oo lb. dry vitamin A Steer number 08-126, a rumen fistulated, 13 month old, 550 1b. Holstein, was placed on each of the three rations two weeks prior to the initial sampling date. The first ration consisted of 18 lb. of second cutting alfalfa hay plus 50 gm. of salt and 5 mg. of cobalt sulfate daily. The second ration consisted of 3 lb. of second cutting alfalfa hay, 12 lb. of T-7 mix, plus 50 mg. of salt daily. The third ration ‘was the same as the second except that 3.26 % urea "262" imas substituted for the 20 % soybean oil meal. Rumen ingesta was obtained from both animals, ventro- tnedial to the fistula plug. The ingesta was chilled in a (jeep-freeze for 30 minutes and then forced through cheese- 47 .39"? 48 cloth to remove the larger feed particles. The smaller feed particles were then removed bypeemmxngthe rumen liquor through glass wool. The rumen liquor was then centrifuged at 1,052 relative centrifugal force. The supernatant was pipetted off for use in the fractionation and determination of physical measurements. The following determinations were performed in duplicate: (1) Five milliliters of supernatant were pipetted into a 500 m1. KJeldahl digestion flask for total soluble nitrogen determination. (2) Twenty-five milliliters of supernatant were pipetted into a 110 m1. volumetric flask for non-heat coagulatable nitrogen determination. Seventy-five milliliters of distilled water were added and the solution boiled for twenty minutes. It was then cooled to room temperature and filtered through Whatman No. 42 filter paper. Twenty-five milliliters of the filtrate were then pipetted into a 500 ml. KJeldahl digestion flask. (3) Twenty-five milliliters of supernatant were pipetted into a 250 ml. pyrex beaker and 100 milliliters of 15% tri- chloroacetic acid added. The mixture was allowed to stand 30 minutes and then filtered through Whatman No. 42 filter paper. Twenty-five milliliters of the filtrate were then pipetted into a 500 m1. KJeldahl digestion flask for tri- chlcroacetic acid nonprotein nitrogen determination. (4) Twenty-five milliliters of supernatant were pipetted into a 250 ml. pyrex beaker and 100 milliliters of 95% ethanol 49 were added. The mixture was then filtered through Whatman No. 42 filter paper. Twenty-five milliliters were then trans- ferred to a 500 ml. KJeldahl digestion flask for alcohol non- protein nitrogen determination. Fifteen milliliters of concentrated sulfuric acid were added to the contents of each digestion flask. Fifteen grams of catalyst (43.75 gm. mercuric oxide plus 1000 gm. sodium sulfate) were added to the flasks. The contents were allowed to digest over a gas flame until 30 minutes after they became clear. The solutions were then cooled to room temperature and 200 milliliters of distilled water were added to each flask. Sixty milliliters of 50% scdium hydroxide solution (contain- ing 125 gm. sodium thiosulfate per 500 gm. of sodium hydroxide) and zinc were then added to each flask and the contents boiled over a KJeldahl electric distillation rack. The distillate was collected in 250 ml. Erlenmeyer flasks containing 10 ml. of 4.5% boric acid, 15 ml. of distilled water, and 5 drops of KJeldahl indicator. The distillations were allowed to proceed until the distillates reached the 125 ml. level in the Erlenmeyer flasks. The distillate-boric acid solution was then titrated to the endpoint with standard 0.02 w. sulfuric acid. The alcohol protein nitrogen fraction and the trichloro- acetic acid protein nitrogen fraction were determined by subtracting the respective nonprotein nitrogen fraction values 50 from the corresponding total soluble nitrogen determinations. Heat coagulatable nitrogen was determined by subtracting non-heat coagulatable nitrogen values from the corresponding total soluble nitrogen determinations. The rumen liquor supernatant was cooled to 10 degrees C. and passed through a modified 25 ml. pipette to determine the flow time. Twenty—five milliliters of the supernatant were bubbled through a frittered glass foammeter to determine the breaking height of the foam column and the stability of the foam. The stability was ranked relatively. RESULTS AND DISCUSSION The main objective of this study was to determine whether specific nitrogen fractions of rumen fluid could be correlated with froth production in ruminants. Total Soluble Nitrogen _--- W The total soluble nitrogen (TSN) found in the supernatant layer of strained, centrifuged rumen fluid obtained from steer number CS-l26 is illustrated in Figure I. The non-froth pro- ducing ration of alfalfa-brome hay resulted in a TSN level below that of the froth producing ration, T-7, but greater than the TSN level of the other froth producing ration contain— ing urea. The latter ration was the same as the T-7 ration, except that urea replaced the soybean oil meal on a nitrogen equivalent basis. Since froth was present when the animal was on either the T-7 or the urea ration, and it was not present when the animal was on the hay ration, it might be concluded that either TSN does not produce frothiness, or that there is a froth inhibiting factor(s) in alfalfa-brome hay. It was found that considerable dirt in the feed manger inhibited froth production. The dirt had been brought in by a burrowing rat. Difficulty was also eXperienced when froth production was attempted without feeding hay. When three pounds of second cutting alfalfa-brome hay were added to the T-7 or urea ration, the animals began frothing in almost all cases. The feeding of cobalt also appeared to aid froth production. 51 _- raw . t .r—" .~ 52 The mean values for the mg.% of TSN for the three rations were as follows: T—7, 93.66 mg.%; hay, 55.99 mg.%; and urea, 54.12 mg.%. Similar information for another animal, C-"07, is repre— sented by Figure II. The TSN level again is greater for the .4 T-7 ration than for the hay ration. The mean mg.w level of TSN for the hay ration was approximately 70% of the mean mg.“ level of the T-7 ration for both animals. The mean mg.% of the TSN was the same for steer C-707 on the hay ration as for steer 08-126 on the urea ration. Frothing was never observed when the hay ration was fed, whereas repeated frothing was observed with the urea ration. The pronounced cycling demonstrated the necessity for sampling at frequent intervals over an extended period of time. _§gnprotein Nitrogen from Trichloroacetic Acid Treatment The nitrogen obtained by treating 100 ml. of centrifuged rumen fluid with 25 m1. of 15% trichloroacetic acid is shown in Figures III and IV. The results have been expressed as mg.% in Figures IIIB and IVE, and as a percentage of the TSN in Figures IIIA and IVA. A study of Figures IIIA and IIIB revealed that the non-froth producing hay ration resulted in a higher level of runiprotein nitrogen (NPN) than did the urea or soybean oil meal froth producing rations. The higher level for the hay was maintained regardless of whether the results were expressed as total mg.% or as a percentage of the TSN (with two exceptions). The NPN level of the urea ration for steer 08-126, eXpressed as mg.%, was much lower than that of the other two rations. 53 However, the level was between that of the hay and soybean oil meal rations when the data were expressed as a percentage of the TSN. The low NPN level, when expressed as mg.%, might be explained by rapid consumption of the nitrogen by the rumen microorganisms. It would be expected that a ruminant on a urea-rich ration would exhibit a high percentage of NPN. This is demonstrated by Figures IIIA and IIIB. The NPN of the urea containing ration was 67% of the TSN, whereas the NPN of the other concentrate ration was only 57% of the TSN. The difference between animals in NPN content of the rumen :fluid, as determined by trichloroacetic acid precipitation, is shown by comparing Figures IIIA and IIIB with Figures IVA Eirnd IVB. The hay ration resulted in the highest level of NPN 1?cxr steer CS-l26, but the lowest level for steer C-707. Thus, NPN determined by trichloroacetic acid precipitation, as shown in Figures IIIA, IIIB, IVA, and IVE, did not appear 13C) significantly influence froth formation and was not accurately measured by trichloroacetic acid precipitation. _;§rotein Nitrogen from Trichloroacetic_gqiddgrga§mgnt The values for the trichloroacetic acid method of protein rui‘131?ogen (PN) determination were obtained by subtracting the tr‘;1-C=hloroacetic NPN values from the correSponding TSN values. Steer CS-126, when receiving the T-7 ration, produced .a higher level of rumen fluid PN than did the animal on either the urea 0p hay ration as revealed in Figures VA and VB. This appeared true he a dless f whether the values were ex ressed as m .% g r o P 8 OP' £2.53 a percentage of the TSN. The feeding of the other froth pro d L'leing ration that contained urea resulted in a level of ‘ 54 PN approximately one—half that of the T-7 ration, but greater than the non-froth producing hay ration. The feeding of the latter ration resulted in a PN level approximately one-third that of the T-7 ration. It was observed that the higher the level of PN, the greater the day to day variation. An inverse relationship for the trichloroacetic acid PN values for steer C-707 as compared to CS-l26 are revealed in Figures VIA and VIB. These limited data show that the PN level resulting from the feeding of the hay ration exceeded the fP-7 ration, regardless of whether the values were plotted as Ing.% PN or as a percentage of the TSN. The "t" test revealed that the trichloroacetic PN values cxf the rumen fluid of these animals, when fed the T-7 ration, vvexre significantly different to the 0.1% level. This was not fir’ue when they were fed the hay ration. Non-heat Coagulable Nitrogen The amount of nitrogen that did not coagulate when rumen fluld was boiled for twenty minutes is shown in Figures VIIA Figure VIIB revealed very erratic lines when the However, and VIIB. nc>1‘An—heat coagulable nitrogen was plotted as mg.%. “flh163r1 these values were plotted as a percentage of the TSN, a. more logical picture was presented. The graph reveals that tIhlEE zoom—froth producing hay ration produced an almost steady line between 95 and 98.5% of the TSN, whereas both the T-7 rat-‘1 on and urea ration lines appear erratic. However, the 111.8,: four observations plotted on the T-7 ration line were Ob tallied when the animal was slightly "off-feed" and pmducmg Ve r53,— Ilittle froth. Conversely, the last four observations ¥ 55 occurred when the animal was frothing heavily. Thus, the line reveals four non-frothing observations with a mean of about 100% of the TSN, and four frothing observations with a mean of about 88% of the TSN. A study of the urea ration line revealed a pattern approx- imating that of the T-7 ration line. The first two and the last four observations occurred when the animal was frothing. The third, fourth, and fifth observations occurred when the animal was either not frothing or only very slightly. The mean of the frothing was approximately 90% of the TSN, and the mean of the tion-frothing observations was approximately 81% of the TSN. It should be noted that the mg.% non-heat coagulable rLitrogen graphs, Figures VIIB and VIIIB, and the TSN graphs, If1;gures I and II, can be almost superimposed upon each other. The non-heat coagulable nitrogen graphs for the rumen -ffi14xid from steer C-707 present a pattern somewhat similar to 1311<>se for steer CS-l26. The non-heat coagulable nitrogen values from the T—7 ration are higher than those from the hay ration, regardless of whether they are plotted as mg.% or as a percentage C>JT' “the TSN. The hay ration produced a more variable picture “El-1311 steer C-707, when plotted as a percentage of the TSN, than ”1 th steer 05-126. Heat Coagulable Nitrogen The heat coagulable nitrogen values for the rumen fluid from steer cs-126, plotted as a percentage of the TSN and as "TE .5%; iheat coagulable nitrogen, are illustrated in Figures IXA and irks. It is apparent that the heat coagulable nitrogen Val lies from the hay ration remain almost constant from one ‘ w m a ’F‘. 56 sampling to the next. The urea and T-7 rations, however, demon- strate variable results that can only be partially explained by studying the behavior of the animal. The graph for the T-7 ration shows the first four values to be low compared with the last four values. The animal's rumen was either not frothing or frothing only very slightly during the first four sampling periods, but was frothing considerably during the last four periods, when the heat coagulable values were much higher. 'When the urea samples were obtained, the animal's rumen contents ‘were exhibiting considerable froth during the first two and Ilast four sampling periods, but either no froth or very little ciuring the third, fourth, and fifth sampling periods. The graphs for the heat coagulable nitrogen of the three 1?Eitions illustrate that when the animal was frothing, the heat coagulable nitrogen values were usually higher and represented 51 ILarger percentage of the TSN, than when the animal was on the n<3n-frothy ration. Heat coagulable nitrogen values for the rumen fluid of stee-r C-707 are plotted in Figures XA and KB. They demonstrate an inverse relationship from those of steer CS-l26. It was noted that, with one exception, when plotted as a percentage (31?. tlhe TSN, the non-froth producing hay ration yielded higher 116351”t= coagulable nitrogen values than the froth producing T-7 I.aLt’-‘-'—On. “Mt—- Nonprotein Nitrogen by Alcohol Precipitation —-—.~-—‘- {The values obtained by precipitating rumen fluid with 95-V % ethyl alcohol are shown in Figures XIA and XIB. The data ‘ 57 display considerable variability between samples from the same ration and also between rations. The froth producing rations generally yielded more NPN, when expressed as a percentage of the TSN, than the non-froth producing hay ration. The NPN values for rumen fluid from steer C-707, which was fed a hay ration, are illustrated in Figures XIIA and XIIB. P] The mean values obtained for NPN from C-707 and CS-l26 were , 27.76 mg.% and 25.74 mg.%, respectively. However, when the (data were expressed as a percentage of the TSN, the difference tmas greater, 50.50% for C-707 and 39.23% for CS-l26. These .9 Vailues were found to be significantly different at the 5% lxevel when the "t" test was applied. Protein Nitrogen by Alcohol Precipitation The PN obtained by precipitating the rumen fluid of steer (353-126 with 95% alcohol is shown in Figure XIIIA. This graph 18 an inverse of Figure XIA, which illustrates the NPN. Thus, V¢11<3r1 the PN is plotted as a percentage of the TSN, the higher ‘VEiiltqes tend to favor the non-froth producing ration. The alcohol precipitated rumen fluid PN for steer 08-125, expressed as mg.%, is shown in Figure XIIIB. The data point (3‘11: ‘bhat the non-froth producing hay ration yielded PN values mldvvay between the values for the froth producing rations. IFigures XIB and XIIIB appear very similar despite the fact that the former figure illustrates the data for NPN and the liitzt3€ézr represents PN. In both graphs, the froth producing T~7 Pa tion containing a high percentage of soybean oil meal Yield ed the greatest amount of rumen fluid nitrogen, the froth DPQ duclng urea ration containing the theoretical equivalent ‘ 58 of nitrogen yielded the least rumen fluid nitrogen, and the non— froth producing hay ration yielded values midway between the two froth producing rations. The alcohol precipitated PN of the rumen fluid from steer C-707, plotted as a percentage of the TSN, is illustrated in Figure XIVA. The mean percentage of the values from the hay ration was 49.50, compared with 59.76 for steer 08-126. The alcohol precipitated PN of the rumen fluid from steer (3-707, expressed as mg.%, is shown in Figure XIVB. The PN Ilevel remained constant, despite the fact that the TSN level \naried considerably. A comparison with the values obtained fWor steer 08-136 on a similar hay ration showed that the rumen 1?]Jdid of steer C—707 yielded 26.41 mg.% PN, whereas steer 08-126 the considerable difference between 371.elded 40.26 mg.%. Thus, animals is again illustrated. Flow Time The seconds required for a specific amount of rumen fluid ‘tCD JfloW'through a graduated pipette is shown in Figure XVA. frfléa results show that the rumen fluid from the T-7 ration reguired the longest flow time, that from the urea ration the '7E31K11: '1ongest, and the rumen fluid from the non-froth producing héltv” Instion the shortest length of time. Thus, the rumen contents "frsc>TT1 the two froth producing rations appeared more viscous than t rlc>$3<>tflo steers are shown in Table I. The levels of significance for the "t" values, obtained t’If‘ comparing the rations for each animal, and by comparing (iii-f‘ferent steers on the same ration, are illustrated in urei‘bfile II. ('31 I80 " my I60 - ISO- I40- 4. ' 1" l30- I20- IIOr Mg. ‘70 IOOP 90'- 80- ’..- ... 7O ” .O."'Io'. ‘ 0' . 0 o ‘ '0 60 __ . ‘ e: s. .. 50- \ / ‘ ” 40' — T-7 ration ---- Hay ration 3%; —— Urea ration o I l I l l l l l l _1 Samples Fig. I. Steer CS-I26, total soluble nitrogen content of rumen fluid '62 IOO' 90*- 80- 70- Mg. ‘70 . —‘ Q 60" ’,¢‘ ‘ ~ ~ "‘.mfl" ..~‘D L o §~~ 50 ‘~ —T-7 ration ~' 4o~ ---- Hay ration O l 1 1 1 .L Samples Fig.11. Sleer C-707, fofol soluble nitrogen content of rumen fluid 90' .. IIII - ‘. 80 )— ..00 .'O'. . -. ..‘.‘ .“ 'C‘. ... ' ' t o‘ 0,0 Of " ‘. . lOl’Ol 70_ I \‘ ' \ soluble \ nitrogen o 50- 50~ — T-7 ration ---- Hay ration 40» —— Urea ration d f l l 1 L l l 1 l 1 l 0 Samples Fig.1HA. Steer C‘s-[26, trichloroacetic acid nonprotein nitrogen content of rumen fluid IOO’ QCM- 80b 70- 6C»- Mg. 0,0 QC...’ 6‘. .G‘ ‘. 50- ‘0’ .0 o“’ 40*- ’~~ w 30b -— T-7 ration 20L ---- Hay ration T -— Urea ration o l L l l J l l l l 1 Samples Fig. 1H 8. Steer C‘s-[26, trichloroacetic acid nonprotein nitrogen content of rumen fluid lOOl' 90- ‘70 Of lota soluble 80‘— nitrogen 70F — T-T ration 60r . ---- Hay ration J oi l J J l J Samphs Fig. Ll? A. Steer C-707, trichloroacetic acid nonprotein nitrogen content of rumen fluid 90 80 70 Mg. °/. 60 50 4O ,- i) ll — T-7 ration ---- Hay ration of L L J L l Samples Fig. 1]? B. Steer 6-707, trichloroacetic acid nonprotein nitrogen content of rumen fluid 7O — T-7 ration 60 '--- Hay ration 5m -— Urea ration ‘70 0f total soluble 40 nitrogen / 30 I ~~" ~‘ ‘~ O § 20 9.... ~~~IIII......--...' .....O 0 l0 1 1 l L l l l l 1 J 0 Samples Fig.1?A. Steer C‘s-[26, trichloroacetic acid protein nitrogen content of rumen fluid 'D‘ ‘V 65 80’ — T-7 ration 70h ---- Hay ration L —— Urea ration 6O ' 50* Mg. ‘70 4a— 30~ ~ \\ ’~ 0 20" V’ \\ o'O .h” '0_ .'O"....~I--.l-Iu=~h ’ ~.§.-.-. l l l l J J I l J J 0 Samples Fig-1733M“ C3426. trichloroacetic acid protein P nitrogen content of rumen flurd . 4° —- T-7 rattan ---- Hay ration 0,0 Of 30- .-‘—.---‘.‘~~~ total . .‘~~ soluble zo- ‘~~~ nitrogen l0- 1 L I l 1 L 0 Samples . . . Fig.1a’A. Steer 6-707, trichloroacetic acid protein 30.. nitrogen content of rumen Tflgid t' — - ra non 20 ---- Hay ration Mg. 0/0 l0- 0 . .P . . . Fig.1! 3. Steer C- 707, trichloroacetic acid protein nitrogen content of rumen flurd IOOl' ’.---..--.-... ‘70 Of ’-- totalI 90.. A solub e a nitrogen " \\ / \ I, . 80— I \ ——T-7 I’OTIOI'I "'H- Hay ration 701E —— Urea ration O 1 1 1 1 j, I 1 i I . Samples . Fig. ETA. Steer CS-l26, non-heat coagulable nitrogen .50_ content of rumen flurd ‘ — T-7 ration l40- "n Hay ratlan -- Urea rattan I30— l20- llO- IOO- Mg. 70 90- 80- O .. " Q 70 . ' - I..' ‘K 0’ .‘S ' ‘ I ‘ 60- \ , s ’ 4o \ / v"'"" ' \ I oTL - L W32). . . , . . Samples . FigJZII B. Steer CS-l26,non-heat coagulable nitrogen content of rumen fImd ‘ I00" -.. 70 Of oO—’--— total 90, soluble ’v' . nitrogen ’,’ — T-7 ration 80,“ .v” ---- Hay ration f (76) o l l l l l Samphs Fig. IZZIZ'A. Steer C- 707, non-heat coagulable nitrogen content of rumen fluid 90' 80- 70- Mg. 70 60— ’o'.~~~ ’¢ ~~~ 50- ," ~‘~~ «’ ‘0 v' . 4o... 0’ — T'7 ratlon ---- Hay ration _ 1 1 1 1 l O . Samples Fig. m B. Steer C-707, non'heat coagulable nitrogen content of rumen fluid 40' — T-7 ration ---- Hay ration 30- % Of -- Urea ration total soluble 20 nitrogen l0 O p Fig.1X A. Steer C‘s-[26, heat coagulable nitrogen content of rumen fluid 4O 30 Mg. 0/o 20 IO —- T-7 ration ---- Hay ration -— Urea ration ~ --,---F=’--- III, Samples. Fig.1X B. Steer 03-126, heat coagulable nitrogen content of rumen fluid 301' :loto: .~ — T-7 ration O 0 -— ~ I.-. ' soluble ~~‘ Hay ration nitrogen IO- 0 Samples Fig.1 A. Steer 0-707, heat coagulable nitrogen content of rumen fluid a 20- — T-7 ration Mg. ’0 '0'- .Q... I.-. Hay ration ...---- o J J Samples Fig. I B. Steer C-707, heat coagulable nitrogen content of rumen fluid 60' 69 ‘70 Of 50- total SOlUble 40" .. nitrogen 30‘ / — T-7 ration ---- Hay ration i —— Urea ration o L L J L J l J L 1 _l Samples Figzr A. Steer CS-l26, alcohol nonprotein nitrogen content of rumen fluid 80' — T-7 ration 70+ ---- Hay ration -— Urea ration 60- 50- Mg. 70 40r 30’ I... * ~ --..o ’ ~.¢~M..-'IO---o 20"- ”\\ / ' I of J L L J l J L J l J Samples Figfl' B. Steer CS-l26, alcohol nonprotein nitrogen content of rumen fluid I 6C) ‘70 Of -——0.~~ fOlOl 50 ....-----‘ §~~~ 33%|; .. --'-Hay ration J ~~‘~. 3L 4 . *1 Samples Fig.211' A. Steer C- 707, alcohol nonprotein nitrogen content of rumen fluid 40F 70 — § 30- .—“" ‘~ Mg. 0’9 ." ~~§~~ ‘~ 20 - . ‘3 ---- Hay ration j 1 I Samples Fig.XZI B. Steer 0-707, alcohol nonprotein nitrogen content of rumen fluid 7o- \\ . o/o Of 60'- total soluble 50- nitrogen — T-7 ration 40: ---- Hay ration j —— Urea ration o I J l L l l I I I l J Samples Figm A. Steer CS-l26, alcohol protein nitrogen content of rumen fluid 90' — T-7 ration 80- ---- Hay ration —— Urea ration 70- 60l- Mg. 0/0 50- VII-.--..Q 40" " ..Q ’ “ . o \ ’0' ‘ ',o .0 \ ’ I... 30F V \\ / o I I I I I J I I I LI Samples Figm B. Steer CS-l26, alcohol protein nitrogen content of rumen fluid 71 60’ ‘70 Of ’,o‘. total __ . "’,.v ol ble "- . :itrtogen ---.--u---QO"‘ i- i: "H" Hay ration El . . L Samples Fig.1!!! A. Steer 0-707, alcohol protein nitrogen content of rumen fluid 30’ Mg o/o gun-n..-Incas-IOIII----------.. 20- . L. ---- Hay ration 0'] L l I Samples Fig. m B. Steer 0-707, alcohol protein nitrogen content of rumen fluid 35" 30- M Sec. 25- m:.=m‘::munuh-n—u¢.=:.... —- T-7 ration ---- Hay ration -- Urea ration I 1 L L Samples Fig.1? A. Steer 05-126, rumen fluid flow time 20 T‘I 04L L l I l ++++ - '0‘ o 0' ~~§ 0' +H” .. o‘Q Q / ‘s 'H' _ — d - h-n - I. — T-7 ration +7 ---- Hay ration —— Urea ration F1. I I I L I I I I I _J 1 Samples l Fig.1? 8. Steer CS-l26, foam stability of rumen fluid .. I I I. so '0. ’- ‘~ 0 S Q 0' ‘ o ' 3 45 " o “ 3 g ' ‘ 0 ‘ I s I s 3 ' “ \ s 35 " I . ao— \ I .25 _ I \ 20 - /\ Mm l5- IO- — T-7 ration 5r- ---- Hay ration —— Urea ration 0 I I I I I I I I L _I Samples Fig.1]? 0. Steer CS-l26, foam height of rumen fluid 73 35" — T-T ration ---- Hay ration 30- Sec. 2 .----------.---------. 5.— 3% . I I Samples Fig. 1121 A. Steer C-707, rumen fluid flow time an, i . .———"— . ~~~~~~~~ l +++~ ‘0 ++ .. M i, + l- _ T'7 ration ---- Hay ration L I I Samples Fig. 233 B. Steer 0-707, foam stability of rumen fluid 35' 30" .-----------O I 25" ” Mm. ’I 20’ h l5" ’I’ . ,’ -— T-7 ration 'Ol' '0’ ---- Hay ration ‘t _ l l I G Samples Fig..m C. Steer 0-707, foam height of rumen fluid 74 TABLE I MEAN VAL ES, STANDARD ERROR, AND NUMBER OF SAMPLES FOR THE CHEMICAL AND PHYSICAL MEASUREMENTS ' OF RUMEN FLUID 2. YR U -o van c:z - wk ~46: r4 HE4 Hco w lo lope to I064 (DE - oa- (Mt O0 Itflh £3 ”38272 "£5.32 ”£15.32. 28' 8813 a E4 z E4 m 24 cu E4 z ::L>z z m Hay Ration Steer C-707 X 54.17 40.74 13.43 75. 9 24.11 48.70 3.2. 5.02 1.79 3.24 3.70 3.76 5.55 N 3 3 3 3 3 3 Steer CS-lf) x 65.99 53.58 12.41 81.20 19.05 54.40 S.E. 2.20 2.03 1.15 1.53 1.16 2.55 N 10 10 10 10 10 9 Urea Ration Steer 08-126 X 54.12 35.84 18.28 66.88 33.12 47.22 3.3. 2.67 1.14 2.04 2.22 2.22 2.57 N 9 9 9 9 9 9 T—7 Ration Steer CS—126 " f X 93.0) 53.29 40.38 57.30 42.39 87.72 8.3. 12.36 7.85 7.52 5.10 5.09 10.24 N 8 8 8 -8 8 8 Steer C-707 f f X 74.40 04.75 9.95 87.08 12.93 72.08 s z 3.21 6.17 3.20 4.12 4.12 5.48 N 5 5 5 5 5 5 X---Mean S.E.--Standard Error of the Mean N-—-Number 0f 81mp1es (Duplicate Analyses for Elch Sample TA BLE I (Continued) 75 0.60 ‘5 - z (DE—10 a) a) E2 15% E3 5) :3 . r-Il H H . El 0) €20 .2: 95 “9:2 255 32 He 3% He 52 Eu 2 :32. 8:32.02 Ewe” o as o oo 24- 22A .4 :30 .1350 50 :21 .c: .2129. .C: .o f: 3 H 4H£h Imam.pmm4 0 c1: 03 o&. -50 HS; 9 (6164-3 Cal-PO ((503430 0:23. 0 04 0| 0U) first: (:5 mo-r-l OO-r—l QOH r-I Hz H94 HZ Hv 730v 4a moz zomemome «2 dm <2 am m 2m ca 5.47 27.76 26.41 50.50 49.50 25.00 22.58 3.50 3.55 4.74 0.40 4.14 4.14 0.29 7.18 0.29 3 3 3 3 3 3 3 25.74 40.26 39.23 59.75 24.75 43.05 2.80 0-74 1-97 1-85 .59 0.13 1.53 0.2: 10 10 10 10 10 10 10 22.99 31.13 42.97 54.62 25.50 18.28 2.11 1.47 2.82 3.01 3.01 0.17 3.04 0.11 9 9 9 9 9 9 9 46.50 54.85 45.38 54.62 28.89 11.64 2.14 8.73 9.23 2.91 2.91 0.48 1.82 0.41 5 5 5 5 7 7 7 _ - - - 29.17 18.23 2.0 - - - - 1 3 0 lyilufll. r.» ‘«t..l. Ililill--. *pfi. 1‘" uzi'. , 'p‘ _v._";.':.-¢z:.‘->w:.’ ”‘2." 7 , 1‘-“ —' TABLE II H a ‘v '7 "I v vs II VI 1" LLVELS OF SIGMIFICAICE USING TELL t TLST gen N acetic Acid Trichloro- acetic Acid NPN Trichloro- acetic Acid PN Trichloro- NPN-% of TSN Trichloro— acetic Acid PN-% of TSN Non-heat Coagulable Nitro T; .. Steer CS-125 Hay Ration VS. H T-7 Ration 5a 1~ 0.10 0.1 5 Hay Ration VS. Urea Rition 1 0.1 \_,'T O H O l--‘ O H T-7 Ration vs. Urea Ration 1 \fl U1 t—‘ Steer 0-707 Hay Ration vs. T-7 Ration 5 1 U‘l Hay Ration Steer 08-125 vs. Steer C—707 5 0.1 \f‘l T—(Eation t er 0 vs. Steer 0-707 1 0.1 0.1 (:1) 126 (J) 3 Significant at the 5% level b Significant at the 1% level C Significant at the 0.1% level 77 2L2 II ( p A T hpAAAnepm maneex :agn - 3.2:. Ki; ‘ 2:2. 30E 2mg ac R an Hogoeaa ‘4 I. r any me m 222 Aegean Em Honooam 2i; Heseefla :we ac m ceaonpflu aafimasmmro pmmm awe mo w :mucppfiz mflanowmoo pwoglcrz cmwcnifiz marmfiswmoo Seem 0.1 0.1 r2 0.1 F) 0.1 0.1 :J 0.1 1 C.) 5 1 ’5’ O 5 m .13 F) _ O. SUMMARY The results of this study to determine whether specific nitrogen fractions of rumen fluid could be correlated with froth production in ruminants have yielded several observations. It was found that there was considerable variability of rumen fluid nitrogen fractions between animals on the same ration. There was also considerable daily variation between rumen fluid Eu .1 m- 7. nitrogen samples. Total soluble rumen fluid nitrogen 223 23 did not appear to influence froth production. The determination of nonprotein and protein nitrogen of rumen fluid by precipitation with trichloroacetic acid did not appear to be a valid method due to incomplete protein precipi- tation. Heat coagulable and non-heat coagulable rumen fluid nitrogen values, when expressed as a percentage of the total soluble nitrogen, were extreme1y variable between both rations and animals and could not be correlated with froth production. The nonprotein nitrogen content of rumen fluid, as deter- mined by an alcohol precipitation method, displayed considerable <20rre1ation with frothing when it was eXpressed as a percentage C)f the total soluble nitrogen. The protein nitrogen content of rumen fluid, as determined tby alcohol precipitation, did not show a positive relationship tlo frothing. 78 L _ 79 Rumen fluid flow time did not appear to be correlated with froth production. Rumen fluid from the non-froth producing ration was found to yield a more stable foam than in the case of froth producing rations. Rumen fluid from the non-froth producing ration also :yielded the greater column of foam when air was bubbled through ‘the liquid. LITERATURE CITED Anonymous. 1952. The Merck Index of Chemicals and Drugs. 6th ed. Merck and Co., Inc. Rathway, N. J. p.498. Anonymous. 1953. What's New in Farm Science. Wis. Agr. Expt. Sta-o, B44111. 5100 pp. 1’40 Ascott, E. w. 1946. Chronic Tympany of the Rumen. Vet. Rec. l 58:39. i Balch, C. C., D. A. Balch, V. w. Johnson, and J. Turner. 1953. j.. Factors Affecting the Utilization of Food by Dairy Cows. j; 7. The Effect of Limited Water Intake on the Digestibility g and Rate of Passage of Hay. Brit. J. Nutr. 7:212-224. ”" Barrentine, B. F., C. B. Shawver, and L. w. Williams. (abs) 1954. Bloat Studies and Observations. J. Animal Sci. 13:1006. Blake, J. T., N. L. Jacobson, and R. S. Allen. (abs 1955. Studies on the Bloat Syndrome. J. Dairy Sci. 3 :606. Clark, R. 1944. The Role of an Adequate Water Supply in the Prevention of Ruminal Impaction in Cattle. J. So. Africa Vet. Med. Assoc. 15:113-119. Clark, R. 1948. Studies on the Alimentary Tract of the Merino Sheep in South Africa XIV. The Effect of Commonly Used Antifermentatives on the In Vitro Formation of Gas in Ruminal Ingesta and Its BEEring on the Pathogenesis of Bloat. Onderstepoort J. Vet. Sci. and Animal Ind. 23:389-393. Clark, R. 1950a. The Paralytic Action of Histamine on the Ruminal Musculature. J. So. Africa Vet. Med. Assoc. 21:13-15. Clark, R. 1950b. The Use of Anti-frothing Agents in the Treatment of Acute Bloat. J. So.Africa Vet. Med. Assoc. 21:173-178. Clark, R. 1951. Studies on the Alimentary Tract of the Merino Sheep in South Africa XX. The Failure of the Ruminal Musculature to Respond to Carbamylcholine-chloride When Paralyzed with Potassium Cyanide. Onderstepoort J. Vet. Research 25:67—72. Clark, R., w. Oyaert, and J. I. Quin. 1951. Studies on the Alimentary Tract of the Merino Sheep in South Africa. XXI. The Toxicity of Urea to Sheep Under Different Conditions. Onderstepoort J. Vet. Research 25:73—78. 80 81 Clark, R.,and J. I. Quin. 1945. Studies on the Alimentary Tract 0f the Merino Sheep in South Africa. XIII. The Role of Prussic Acid in the Aetiology of Acute Bloat. Onderste- poort J. Vet. Sci. and Animal Ind. 20:209-212. Clark, R., and K. E. Weiss. 1952a. Factors Contributing Towards Bloat in Ruminants. J. 80. Africa Vet. Med. Assoc. 23: 103-106. Clark, R., and K. E. Weiss. 1952b. Reflex Salivation in Sheep and Goats Initiated by Mechanical Stimulation of the h_ Cardiac Area in the Fore Stomachs. J. 80. Africa Vet. Med. 2 Assoc. 23:163-165. 3 l Cole, H. H., R. S. Amadon, R. W. Dougherty, Dwight Espe, Carl 5 F. Huffman, T. M. Olson, and A. F. Shalk. 1943a. Recom- i mendations for Prevention of Bloat in Cattle and Sheep. J. Am. Vet. Med. Assoc. 103:294-296. Cole. H. H.. C. F- Huffman, M. Kleiber, T. M. Olson, and A. F. 4- Schalk. 1945. A Review of Bloat in Ruminants. J. Animal 8°10 4:183-236o Cole, H. R., C. F. Huffman, Max Kleiber, R. E. Hungate, W. D. Maclay, C. R. Thompson, R. W. Daugherty, H. W. Colwin Jr. 1955. A Review of Bloat in Ruminants. (In Press) Cole, H. R., and Max Kleiber. 1945. Bloat in Cows on Alfalfa Pasture. Am J. Vet. Research 6:188—193. Cole, H. H., and Max Kleiber. 1948a. Studies Bearing on the Bloat Problem. (abs). J. Dairy Sci. 31:668. Cole, H. H., and Max Kleiber. 1948b. Studies on Ruminal Gas Formation and the Consumption of Alfalfa Pasture by Cattle. J. Dairy Sci. 31:1016-1023. Cole, H. H., and S. W. Mead. 1943. A Physical Deficiency in the Ration of Ruminants. Science 98:543—544. Cole, H. R., S. W. Mead, and M. Kleiber. 1942. Bloat in Cattle. Calif. Agr. EXpt. Sta., Bull. 662. Cole, H. H., S. W. Mead, and W. M. Regan. 1943b. Production and Prevention of Bloat in Cattle on Alfalfa Pasture. J. Animal Sci. 2:285-294. (Jolvin, H. W. Jr., P. T. Cupps, and C. R. Thompson. 1955. The Effect of Alfalfa Saponin 0n Rumen Activity in Sheep. (abs). J. Dairy Sci. 38:606. Ikougherty, R. w. 1940. Physiological Studies of Induced and Natural Bloat in Dairy Cattle. J. Am. Vet. Med. Assoc. 96:43—46. 82 Daugherty, R. W. 1941. Further Investigations of Rumen Gases and Bloat in Ruminants. J. Am. Vet. Med. Assoc. 99:110-114. Daugherty, R. W. 1942a. Analysis of Gas and Ingesta of Bloated Cattle. J. Am. Vet. Research 3:401-402. Daugherty, R. W. 1942b. A Study of Drugs Affecting the Motility of the Bovine Rumen. Cornell Vet. 32:269-280. Daugherty, R. W. 1953. The Continuing Quest for the Cause of Bloat in Ruminants. J. Am. Vet. Med. Assoc. 122:345-353. Daugherty, R. W., and R. M. Cello. 1949. A Study of Toxic Factors in the Rumen Ingesta of Cows and Sheep. Cornell Daugherty, R. W., and Robert M. Cello. 1952. Studies of the Toxic Factors in Rumen Ingesta of Cows and Sheep. II. . Am. Vet. Med. Assoc. 89th. Annual Meeting. pp. 130-137. 1 7 Daugherty, R. W., and R. B. Christensen. 1953. In Vivo Ab- sorption Studies of Hydrocyanic Acid of Plifit Juice Origin. Cornell Vet. 3:481-486. Daugherty, R. W., and Courtney D. Meredith. 1954. Antifoaming (defrothing) Agents in the Treatment and Prevention of Bloat. J. Am. Vet. Med. Assoc. 124:474-475. Daugherty, R. W., and Courtney D. Meredith. 1955. Cinefluoro— graphic Studies of the Ruminant Stomach and 0f Eructation. Am. J. Vet. Research 16:96-100. Daugherty, R. W., Courtney D. Meredith, and Robert B. Barrett. 1955. Physiological Effects of Insufflation of the Stomach of Sheep. Am..J. Vet. Research 16:79-90. Duckworth, J., and W. Godden. 1939. The Partition of Serum Calcium in Domestic Animals-Some Pathological Cases. Vet. J. 95:386-392. Dukes, H. H. 1947. The Physiology of Domestic Animals, Comstock PUbliShing COO, Inoc’ ItKa-ca’To YT, F‘s 329. Espe, Dwight, and C. Y. Cannon. 1940. Some Factors Relating to Bloat in Cattle. (abs). J. Dairy Sci. 23:550. IEspe, Dwight, Norman L.Jacobson, and Clawson Y. Cannon. 1943. Why Do Cows Bloat? Holstein-Friesian World. 40:1020,1056. Evans, W. C., and E. T. R. Evans. 1949. Relation of a Clover Juice Factor Causing Paralysis of Isolated Muscle to Bloat in Ruminants. Nature 103:373-377. I”(Erguson W. S. 1948a. A Factor in Pastur s Affe t scl Activity. Grassland Conf. Handbook. 7Creat firi92143. e 83 Ferguson, W. S. 194%b. A Possible Relation Between the Action of Clover Juice on Isolated Muscle and ”Bloat" in Cattle. Nature 131:810-817. Ferguson, W. S., De B. Ashworth, and R. A. Terry. 19A9. Nature of a Musc1e~inhibiting Compound in Lucerne and_its Possible Connection with Bloat in Cattle. Nature 1a3zaOo-007. Ferguson, W. S., De B. Ashworth, and R. A. Terry. 1950. Identity of a Muscle- inhibiting Flavone in Lucerne. Nature la6: 115-117. Hale, W. R., and R. P. King. 1955. Possible Mechanism of Urea Toxicity in Ruminants. Exp. Biol. and Med. 89:112—114. Hancock, John. M9 )4 Studies in Grazing Behavior of Dairy Cattle. II. Bloat in Relation to Grazing Behavior. J. Agr. Sci. 45:80-95. Heath, D. F., and P. 0. Park. 1953 An Irreversible Choline- esterase Inhibitor in White Clover. Nature 172:200. Henrici, M. 1952 . Comparative Study of the Content of Starch and Sugars of Tribulus terrestris, Lucerne, some Gramineae, and Pentzia incana Under Different Meteorological, Edaphic, and Physiological Conditions. II. Carbohydrate Nutrition (Bloating and Dikkap Sickness). Onderstepaort J. Vet. Research %5 45- 92. Houssay, Bernardo A., Jean T. Lewis, Oscar Orias, Eduardo Braun Menendez, Enrique Hug, Virgilio G. Faglia, and Luis F. Lelair. 1951. Human Physiology lst. ed., McGraw Hill Book Co., Inc. New York, N. Y., p. 45. Huffman, C. F. 19 5. Personal Communication. Hungate, R. E., R. W. Daugherty, M. P. Bryant, and R. M. Cello. 1952. Microbiological and Physiological Changes Associated With Acute Indigestion in Sheep. Cornell Vet. 42:A23-449. Jacobson, Dan R., and Ivan L. Lindahl. 1955. Studies on Bio- chemical, Physical, and Bacteriological Factors Involved in Feed Lat Bloat. Md. Agr. Expt. Sta., Misc. Pub. 238, pp. 9‘15. ‘Iacabson, Norman L., Dwight Espe, and C. L. Cannon. 1942. Factors Modifying the Rate of Fermentation of Rumen In- gesta and Their Possible Relation to Bloat in Dairy Cattle. J. Dairy Sci. 25:785-799. JXDhns, A. T. 1954. Bloat in Cattle on Red Clover I. N. Z. J. Sci. and Tech. 35:289-321 84 Kerr, W. R. ., and H. G. Lamont. 1945. Haven - an Expression of /' Allergy. Vet. Rec. 58:3-7. Kleiber, Max, H. H. Cole, and S. W. Mead. 19’13. Bloat in Cattle and Composition of Rumen Gases. J. Dairy Sci. 26:929-933 Knapp, E., A. L. Baker, and T. W. Phillips. 1943. Variations in the OCcurrence of Bloat in the Steer Progeny of Beef Bulls. J. Animal Sci. 2:22 -225. Kamarov, S. A., and G. W Stavraky. 1940. The Nitrogenous Constituents of Cat's Submaxillary Saliva Evoked by Para- sympathetic and Sympathetic Stimulation. Can. J. Research 18D:233-247. Lamont, H. G. 1943. Haven - an Expression of Allergy. Vet Rec 58: :2 _ro3 Lindahl, Ivan L., A. C. Cook, R. E. Davis, and W. D. Maclay. 1954. Preliminary Investigations on the Role of Alfalfa Saponin in Ruminant Bloat. Science 119:157-158. Lindahl, Ivan L., and Russell L. Davis. 1954. Some Factors in Feed Lat Bloat (abs). J Animal Sci. 13:1024. McGilliard, A. D. 1955. Personal Communication. McIntosh. R. A. 1941. Digestive Disturbances of Cattle. J. Am. Vet. Med. Assoc. 98:441-447. Mead, S. W., H. H. Cole, and W. M. Regan. 1944. Further Studies on Bloat. J. Dairy Sci. 27:779-791. Newbold, R. P. 1954. Preliminary Note on the Extraction of a Bloat-promotin Fraction From Red Clover. N. Z. J. Sci. and Tech. 36:2 5-285. Nichols, R. E. 1951. A Note an Eructatian in Sheep as Related to Rumen Fluid Level and Gas Insufflation. Am. J. Vet. Research 12:199-200. Nichols, R. E., W. E. C. Moore, and R. D. Dillon. 1955 The Effective Buoyancy of the Rumen Juice of Cattle Fed Hay, Grass, and Fresh Legumes. J. Animal Sci. 14: -276- 278. Olson, T. M. 1940a. Bloat in Dairy Cattle. So. Dak. Agr. Expt. Sta. Circ. 27. pp. 1-8. Olson, T. M. 1940b. Bloat in Dairy Cattle. J. Dairy Sci. 343-353. Olson, T. M. 1 OAOC. Bloat in Dairy Cattle. J. Dairy Sci. (abs). 23. Al 2-A123 [0 U) 85 Olson, T. M. 1942. Ruminal Gases in Normal and Bloated Animals. J. Dairy Sci. 25: 584-085. Olson, T. M. 1944. Bloat in Dairy Cattle. So. Dak. Agr. Expt. Sta., Circ. 52. pp. 1-11. Parsons, A. R., Glen Hall, and W. E. Thomas. 1952. Forage Extracts Which Cause Bloat or Death When Administered Orally to Sheep. J. Animal Sci. 11:772-773. Parsons, A. R., A. L. Neumann, C. K. Whitehair, and Jesse Sampson. 1955. Isolated Cut and Rumen Motility as Affected by Extracts From Bloat Producing Forages. J. Animal Sci. 14:403-411. -¢- _-‘————w-c—_ '4 .v- — Parsons, A. R., C. K. Whitehair, and A. L. Neumann. 1953. 5 Bloat and Rumen Motility as Affected by Forage Extracts and i 1 Other Related Substances. J. Animal Sci. 12. 926- 927. pl 1 ‘1‘” l' W“” Petersen, W. E. 1945. (Cited by Cole, H. H., C. F. Huffman, M. Kleiber, T. M. Olson, and A. F. Schalk. 1945. A review of Bloat in Ruminants. J. Animal Sci. 4:211). Petersen, W. E. 1950. Dairy Science. 2nd. ed. J- B. Lippincott Co., Chicago, Ill. p. 419. Petersen, W. E. 1955. Personal Communication. Potter, George C. ., and Fred A. Kummerow. 1954. Chemical Simi- larity and Biological Activity of the Saponins Isolated from Alfalfa and Soybeans. Science 12 0: 224-2 25. Quin, A. H., J. A. Austin, and Karl Ratcliff. 1948. A New Approach to the Treatment of Bloat in Ruminants. J Am. Vet. Med. Assoc. 114. 313— 314. Quin, J. 1943. Studies on the Alimentary Tract of the Merino Sheep in South Africa VIII. The Pathogenesis of Acute Tympanities. Onderstepoart J. Vet. Sci. and Animal Ind. 18:113-117. Quin, J. I., W. Oyaert, and R. Clark, 1951. Studies on the Alimentary Tract of the Merino Sheep in South Africa XVIII. The Effect of Fasting an the Activity of the Ruminal Flora of Sheep and Cattle. Onderstepoart J. Vet. Research 25:51-58. Quin, J. 1., and J. G. Van Der Wath. 1938. Studies on the Alimentary Tract of the Merino Sheep in South Africa V. The Motility of the Rumen Under Various Conditions. Onderstepaort J Vet. Sci. and Animal Ind. 11:361—370. .,_._.H__.._ W‘. 86 Reed, Howard S., and Jean Dufrency. 1942. Catechal Aggregates in the Vacuoles of Cells of Zinc Deficient Plants. Am. J. Bot. 29:544-551. Regan, W. M. and S. W. Mead. 1945. Unpublished Data. (Cited by Cole, H. H., C. F. Huffman, Max Kleiber, T. M. Olson, and A. F. Schalk. 1945. A Review of Bloat in Ruminants. J. Animal Sci. 4:215). Repp, Ward W., W. H. Hale, E. W. Cheng, and Wise Burroughs. 1955. The Influence of Oral Administration of Nanprotein Nitrogen Feeding Compounds Upon Blood Ammonia and Urea Levels in Lambs. J. Animal Sci. 14:118-131. (Cited by Hale, W. H. and R. P. King. 1955. Exp. Biol. and Med. 89:112-144.S Shanks, P. L. 1946. Bloat. J. Ministry Agr. 53:53-55. Smith, C. K. 1954. Personal Communication. Smith, C. K., J. R. Brunner, C. F. Huffman, and C. W. Duncan. 1953. Experimental Production of Frathy Bloat in Cattle. J. Animal Sei- 12:932. Smith, C. K., and R. S. Emery. 1955a. Unpublished Data. Mich. Agr. Expt Sta. Smith, C. K., and R. S. Emery. 1955b. Personal Communication. Stone, Ernest C. 1949. Fermentation Ability of Ingesta from Normal and Atonic Bovine Rumens. Am. J. Vet. Research 10:26-29. ‘ . -Sumner, James B., and G.Fred Somers. 1943. Chemistry and Methods of Enzymes. Academic Press, Inc., New-York, N. Y. EEI‘II9.292. Weiss, K. E. 1952. Physiological Studies in Eructation Reaction in Relation to Bloat in Cattle. Proc., Sixth Int. Grass- land Cong. 2:1235-1236. ‘weiss, K. E. 1953a. The Significance of Reflex Salivation in Relation to Frath Formation and Acute Bloat in Ruminants. Onderstepaart J.Vet. Research 26:241-250. VVeiss, K. E., 1953b. Physiological Studies on Eructation in Ruminants. Onderstepoort J. Vet. Research 26:251—283. . as on: {VLAJUSH Demco-293 ST A ”7111111717le MIMI Will] IE Mil/W”