AMMONIUM SALTS OF 0me was AS SOURCES or CRUDE Pmmu ~ FOR FEEDLOT emu: Thesis for the Degree of Ph. D.. MICHIGAN STATE UNIVERSITY CHARLES KELLER ALLEN 119172 This is to certify that the thesis entitled Ammonium Salts of Organic Acids as Sources of Crude Protein for Feedlot Cattle presented by Charles Keller Allen has been accepted towards fulfillment of the requirements for __P_ml3_.__degree in _Animal_nusbandry 111”; ‘.’.!’L ’91-:AA Date September 13: 1972 0-7639 S’ ‘ \3 BINDING BY ' IIIIAG & SIIIIS' 800K BINDERY IIIIZ. LIBRARY BINDERS ABSTRACT AMMONIUM SALTS OF ORGANIC ACIDS AS SOURCES OF CRUDE PROTEIN FOR FEEDLOT CATTLE BY Charles Keller Allen The feeding value, metabolism and utilization of supplemented organic acids or ammonium salts of organic acids were studied in four feeding trials and three meta- bolic studies. The effect of high versus moderate levels of concentrate on the utilization of ammonium salts was also studied. In Experiment I, ammonium acetate, ammonium lactate, soybean meal and urea were compared in a growth study on a ration composed of 75 per cent concentrates and 25 per cent corn silage. Cattle supplemented with ammonium acetate gained 5.4 per cent faster than cattle receiving soybean meal and 12 per cent faster (P<.05) than urea fed steers. Ammonium lactate feed steers gained faster (P<.05) than soybean meal (13.5 per cent) or urea (20 per cent) fed groups. Consumption was increased 9.1 per cent and 12.2 per cent for the ammonium lactate steers and 3.1 per cent and 6.0 per cent for the ammonium acetate fed steers compared to those fed soybean meal and urea, Charles Keller Allen respectively. Similarly, feed efficiency was increased 3.5 per cent and 7.2 per cent with supplementation of ammonium lactate and 2.3 per cent and 6.1 per cent when ammonium acetate was fed. In Experiment II, ammonium salts of formic, acetic, propionic lactic and butyric acids were compared to soy- bean meal, urea and a supplement that derived one-half of its protein equivalent from urea and one-half from corn steep water (U-CSW). The nitrogen supplements were added to either a ration of 40 per cent concentrates and 60 per cent corn silage or a ration of 80 per cent concen- trates and 20 per cent corn silage. None of the differences in gains were significant. However, the urea fed cattle had the lowest gains, feed efficiency and DM consumption and produced carcasses that had the lowest grade and the least fat. The performance of all NPN fed cattle except those fed ammonium acetate increased with a higher level of concentrate in the ration. The groups fed NPN were also more efficient on the high concentrate diet but less efficient on the low concentrate diet than those fed soybean meal. The de- creasing order of commercial value for the nitrogen supplements was ammonium propionate, soybean meal, ammonium butyrate ammonium lactate, ammonium formate, U-CSW, ammonium acetate and urea. Charles Keller Allen The gains of cattle on high concentrate diets were 2.9 per cent higher and the feed efficiency was 7 per cent greater than on low concentrate diets. However, the low concentrate ration resulted in more beef per hectare, higher gross returns per hectare and lower cost of gains. Cattle fed the high concentrate diet had higher cracass grades (P < .05) and dressing percents (P < .01) and were fatter (P < .01) than those fed the low concentrate diet. Experiment III was designed to study the addition of lactic or acetic acid in equalmolar concentrations as the ammonium salts fed in Experiment I. The acid additions were compared on both urea and soybean meal supplemented rations. Adding acetic acid decreased feed efficiency 3.7 per cent but adding lactic acid increased feed ef- ficiency 2.6 per cent. The addition of lactic acid tended to reduce the amount of carcass fat. Urea fed cattle had a decrease of 4.3 per cent, 2.6 per cent and 1.9 per cent in gain, consumption and feed efficiency, respectively, when compared to soybean meal fed steers. The urea supplemented steers were also trimmer (p < .05) than steers fed soybean meal. In Experiment IV, the addition of acetic or lactic acids to all silage rations was studied. The addition of acetic acid to the ration depressed gain and feed efficiency for all levels of acetic acid. Average daily gain feed Charles Keller Allen efficiency and consumption were not affected by adding lactic acid to the ration. Experiment V involved stomach pumping and bleeding cattle previously on Experiments I and III. The rumen ammonia concentrations were highest at T2.5 for trea, ammonium acetate and ammonium lactate. Ammonium lactate fed cattle also had higher (P < .05) rumen ammonia levels at T and T 5 10' cattle fed ammonium salts than for cattle fed urea or Blood urea levels were higher at all for soybean meal at all sampling times. Rumen acetate concentration was higher at all determinations for cattle fed ammonium lactate than for cattle receiving ammonium acetate or urea and higher for steers fed ammonium salts at T2 than for those fed soybean meal or urea. Rumen propionate levels were higher for the steers fed soybean meal and ammonium lactate. Rumen butyrate was not detected in rumen fluid samples of the ammonium lactate fed steers. When lactic acid was added to the ration, rumen ammonia and blood urea levels were higher at all sampling times. Cattle receiving acetic acid in the diet had the highest rumen acetate at T2.5. Lactic acid fed steers had the highest rumen acetate (except T2.5) and highest rumen propionate at all sampling times. Charles Keller Allen In Experiment VI, nitrogen balance and metabolic studies were conducted with fistulated steers fed the same rations as Experiment I. Rumen ammonia and blood urea levels of the urea fed steers were higher at all sampling times except T10 than for soybean meal or ammonium salt fet cattle. Supplementation with ammonium lactate in- creased the levels of rumen acetate and propionate. Rumen butyrate levels were higher for steers feed ammonium salts than for steers fed urea or soybean meal. Steers fed ammonium salts had greater nitrogen intake but did not lose any more nitrogen in the feces or urine than the soybean meal and urea fed steers. This resulted in an increased quantity of nitrogen digested and higher (P < .01) nitrogen balance for the steers fed ammonium salts. Experiment VII was a nitrogen balance and meta- bolic study of the nitrogen supplements fed in Experiment II on the low concentrate ration. Rumen ammonia concen- trations were maximized at T2 and were highest for ammonium lactate and ammonium acetate fed steers. There was no significant difference among the various nitrogen sources tested for blood urea levels. Rumen acetate concentrations were higher for am- monium acetate fed steers at T2 but lower than soybean meal, ammonium lactate, U-CSW, ammonium formate and ammonium butyrate at T4. Supplementing the diets with soybean Charles Keller Allen meal or ammonium lactate resulted in elevated rumen pro- pionate levels. Rumen butyrate was higher at T T4 and 2! T6 for the ammonium butyrate fed steers. None of the differences in DM intake, DM digesti- bility or the nitrogen parameters studied were significant. Daily nitrogen intake was highest for the U-CSW fed steers, lowest for the soybean meal steers and intermediate for cattle fed urea or ammonium salts. The results of these experiments indicate that ammonium salts of organic acids are superior to urea and in many cases equal or superior to soybean meal in pro- moting efficient beef gains and positive nitrogen balance. The acid portion of ammonium salts is an efficient energy source and does not, with the exception of ammonium acetate, effect DM consumption. AMMONIUM SALTS OF ORGANIC ACIDS AS SOURCES OF CRUDE PROTEIN FOR FEEDLOT CATTLE BY Charles Keller Allen A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1972 Charles Keller Allen Candidate for the degree of Doctor of Philosophy DISSERTATION: Ammonium Salts of Organic Acids as Sources of Crude Protein for Feedlot Cattle OUTLINE OF STUDIES: Major Area: Animal Husbandry (Ruminant Nutrition) Minor Subject: Biochemistry BIOGRAPHICAL ITEMS: Born: December 15, 1942; Independence, Virginia Undergraduate Studies: Virginia Polytechnic Institute, 1965-1969 Graduate Studies: Michigan State University, 1969-1972 MEMBER: American Society of Animal Science Alpa Zeta Phi Kappa-Phi National Block and Bridle Club ii . ‘—-_ AC KNOWLEDGMENT S The author extends his appreciation to Dr. Hugh E. Henderson for his valued guidance and counsel during his graduate program. The author is also indebted to Dr. William T. Magee, Dr. J. T. Huber and Dr. Richard W. Luecke, as members of his graduate committee, for their sound advice and participation in his graduate program. The author also wishes to thank Dr. Ronald H. Nelson and Dr. J. A. Hoefer for making the facilities of Michigan State University and the Michigan Agricultural Experiment Station available for this research. Appreciation is also extended to Dr. Werner G. Bergen for his advice and assistance in interpretation of laboratory results and to Mrs. Phyllis A. Whetter for her assistance in laboratory analysis. The author is grateful to his parents for their support and encouragement. iii TABLE OF CONTENTS Page LIST OF TABLES O O O O C O O O O O O 0 ix Chapter I. INTRODUCTION . . . . . . . . . . . 1 II. LITERATURE REVIEW . . . . . . . . . . 6 Historical DevelOpment of Protein Nutrition . . . . . . . . . . . 6 Nitrogen Metabolism in the Ruminant . . . 8 Post-ruminal Metabolism of Protein. . . . ll Amino Acid Supply and Requirements of Ruminants . . . . . . . . . . 12 Urea as a Nitrogen Source in Ruminants . . 15 Urea as an Additive to Corn Silage. . . . 21 Urea Utilization, Ammonia Absorption and Urea Toxicity . . . . . . . . 24 Other Non-protein Nitrogen Compounds . . . 25 Pro-Sil Addition to Corn Silage. . . . . 30 monium salts O O O C O C O O O O 33 Utilization of Volatile Fatty Acids . . . 39 The Effect of Organic Acid Additions on Intake . . . . . . . . . . . 44 sumary I O O O O O O O O O O O O 47 III. MATERIALS AND METHODS. . . . . . . . . 49 Experiment I--Feeding Trial Comparing Ammonium Acetate, Ammonium Lactate, Urea and Soybean Meal . . . . . . . 49 Design. . . . . . . . . . . . 49 Harvesting of Feed. . . . . . . . 49 Production of Ammonium Salts . . . . 50 Feed Analysis . . . . . . . . . 50 Feeding Trial . . . . . . . . . 51 iv Chapter Experiment.II-—Feeding Trial Comparing Several Ammonium Salts of Organic Acids with Conventional Nitrogen Supplements. . . . . . . . . Design. . . . . . . . . . Harvesting of Feed. . . . . . Production of Nitrogen Supplements Feed Analysis . . . . . . . Feeding Trial . . . . . . . Experiment III--Acetic and Lactic Acid Addition to a High Concentrate Rations O O O I O O O O O 0 Design. . . . . . . . . . Harvesting of Feed. . . . . . Feed Analysis . . . . . . . Feeding Trial . . . . . . . Experiment IV--Acetic and Lactic Acid Additions to all Silage Rations . . Design. . . . . . Harvesting of Feed. . Feed Analysis . . Feeding Trial . . Experiment V--Study of Rumen and Blood Parameters Resulting from Addition of Ammonium Salts or Organic Acids to Cattle Rations. . . ._ . . . . Design. . . . . . . . Management . . . . Sample Collection . . Laboratory Analysis . Experiment VI--Nitrogen Balance Study with Ammonium Acetate, Ammonium Lactate, Urea and Soybean Meal . . . . . . . Design. . . . . . Feeding Regime . . . Sample Collection . . Laboratory Analysis . Page 56 56 56 57 59 60 63 63 65 65 65 67 67 67 67 67 68 68 68 69 7O 72 72 72 74 75 Chapter Experiment VII--Nitrogen Balance Study Comparing Various Ammonium Salts of Organic Acids With Conventional Nitrogen Supplements. . . . . . . Design. . . . . . Feeding Regime . . . . Sample Collection . . Laboratory Analysis . Statistical Analysis . . . . . . . IV. RESULTS AND DISCUSSION . . . . . . . Experiment I--Feeding Trial Comparing Ammonium Acetate, Ammonium Lactate, Urea and Soybean Meal . . . . . . Cattle Performance. . . . . . . Relative Value of Supplements . . . Carcass Evaluation. . . . . . . Experiment II--Feeding Trial Comparing. Various Ammonium Salts of Organic Acids with Conventional Nitrogen Supplements. Cattle Performance. . . . Relative Value of Supplement Carcass Evaluation. . . . Concentrate Levels. . . . Experiment III--Acetic and Lactic Acid Additions to High Concentrate Rations . Cattle Performance. . . Relative Value . . . . Carcass Evaluation. . . Soybean Meal vs. Urea. . Experiment IV--Acetic and Lactic Acid Additions to all Silage Rations . . . Acetic Acid Addition . . . . ._ . Lactic Acid Addition . . . . . . vi Page 75 75 77 78 78 79 80 80 80 84 85 85 85 93 94 96 97 _ 97 103 104 104 106 106 108 Chapter Page Experiment V--Study of Rumen and Blood Parameters Associated with the Addition of Ammonium Salts or Organic Acids to Cattle Rations. . . . . . . . . . 110 Rumen Ammonia and Blood Urea Concen- trations for Nitrogen Sources . . . 110 Rumen VFA Concentrations for Nitrogen Sources. . . . . . . . . . . 112 Rumen Ammonia and Blood Urea Concen- trations for Acid fed Cattle. . . . 114 Rumen VFA Concentrations for Acid Fed Cattle . . . . . . . . 114 Rumen Ammonia and Blood Urea Concen- trations on Soybean Meal and Urea Rations. . . . . . . . . . . 116 Rumen VFA Concentrations on Soybean Meal and Urea Rations . . . . . . 119 Experiment VI--Nitrogen Balance Study with Ammonium Acetate, Ammonium Lactate, Urea and Soybean Meal . . . . . . . . . 119 Rumen Ammonia and Blood Urea Concen- trations . . . . . ._ . . . . 119 Rumen VFA Concentrations. . . . . 121 Dry Matter Intake and Digestibility . . 123 Nitrogen Balance . . . . . . . . 125 Experiment VII--Nitrogen Balance Study Comparing Various Ammonium Salts of Organic Acids with Conventional Nitrogen Supplements. . . . . . . . . . . 125 Rumen Ammonia Concentrations . . . . 125 Blood Urea Concentrations . . . . . 128 Rumen Acetate Concentrations . . . . 129 Rumen Propionate Concentrations . . . 131 Rumen Butyrate Concentrations . . . . 133 Dry Matter Intake and Digestibility . . 135 Nitrogen Balance . . . . . . . . 135 v. GENERAL DISCUSSION. . . . . . . . . . 139 VI. SUMMARY . . . . . . . . . . . . . 144 BIBLIOGRAPHY . . . . . . . . . . . . . 153 vii Chapter APPENDICES I. II. PRE-EXPERIMENT PERFORMANCE. . EXPERIMENT V: MEANS OF INDIVIDUAL PROTEIN-ACID TREATMENT GROUPS viii Page 179 183 LIST OF TABLES Table Page 1. Metabolic response to ammonium salts . . . . 35 2. Comparison of selected ammonium salts. . . . 36 3. Experiment I: Average chemical analysis of feeds. . . . . . . . . . . . . 52 4. Experiment I: Composition of rations fed . . 54 5. Experiment I: Ingredient composition of supplements used . . . . . . . . . . 55 6. Experiment II: Composition of supplements . . 58 7. Experiment II: Ingredient composition of urea - corn steep water supplements. . . . 58 8. Experiment II: Average chemical analysis of feeds fed . . . . . . . . . . . 51 9. Experiment II: Ration dempositium on a DM baSiSo I O O I O I O O I O O O O 62 10. Experiment II: Formulation of mineral supplement . . . . . . . . . . . . 64 ll. Experiment III: Per cent ration composition on a DM basis . . . . . . . . . . . 66 12. Experiment IV: Formulation of 714 supplement . 69 13. Experiment IV: Per cent ration composition on a DM basis . . . . . . . . . . . . 70 14. Experiment VI: Metabolic study treatments 3 utilized 0 O O I O I O O O O O 0 7 15. Experiment VI: Design of experiment . . . . 73 16. Experiment VII: Design of experiment. . . . 76 ix Table 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. Experiment I: Ammonium salts as a source of crude protein for feedlot cattle. . . Experiment II: Ammonium salts as a source of crude protein for feedlot cattle. . . Experiment II: Effect of various crude protein sources and concentrate levels. . Experiment II: 40% shelled corn and 60% corn silage vs. 80% skilled corn and 20% corn silage . . . . . . . . . . Experiment III: Effect of acetic acid and lactic acid addition. . . . . . . . Experiment III: Acetic acid and lactic acid addition to soy and urea supplemented rations O O O O O O O O I O O I Experiment III: Soybean meal vs. urea supple- ments 0 O I C O O O O O O O O 0 Experiment IV: Acetic acid addition to all silage rations. . . . . . . . . . Experiment IV: Lactic acid addition to all silage rations. . . . . . . . . . Experiment V: Mean rumen ammonia and blood urea values for steers fed ammonium salts. Experiment V: Mean rumen VFA concentrations for steers fed ammonium salts. . . . . Experiment V: Mean rumen ammonia and blood urea values for acid fed steers . . . . Experiment V: Mean rumen VFA concentrations for acid fed steers . . . . . . . . Experiment V: Mean blood and rumen values for urea and soybean meal supplemented animals Experiment VI: Mean rumen ammonia and blood urea values. . . . . . . . . . . Page 81 86 89 98 99 101 105 107 109 111 113 115 117 118 120 Table Page 32. Experiment VI: Mean Rumen VFA concen- trations. . . . . . . . . . . . . 122 33. Experiment VI: Effect of nitrogen sources on digestion parameters. . . . . . . . 124 34. Experiment VII: Mean rumen ammonia concen- trations. . . . . . . . . . . . . 126 35. Experiment VII: Mean blood urea concen- trations. . . . . . . . . . . . . 127 36. Experiment VII: Mean rumen acetate concen- trations o o o o I o o o o o o o o 130 37. Experiment VII: Mean rumen propionate concentrations. . . . . . . . . . . 132 38. Experiment VII: Mean rumen butyrate concen- trations. e o o o e o o o o o o o 134 39. Experiment VII: Mean rumen pH values. . . . 136 40. Experiment VII: Effect of nitrogen sources on digestions parameters . . . . . . . . 137 xi INTRODUCTION Recent studies indicate that if present relative rates of population growth continue, it is probable that the excess of cereal grains and natural proteins, which are now available for use as animal feeds, will be critically diminished. More efficient utilization of natural protein and cereal grains can be made if they are consumed di- rectly by man instead of being processed into meat by animals. Only animals which are the most efficient in the utilization of these feeds will be used to convert them into more nutritionally complete food. In this re- spect, ruminants are the least efficient of farm animals and beef cattle are the least efficient ruminants that are in commercial production. However, ruminants survive and are economical when fed materials that are non-edible by man and other monogastrics. This results from a digestive system which includes a symbiotic relationship with a massive microbial population capable of digesting fibrous plant material that is high in cellulose and not digestible by simple stomach animals. The final products of this digestive process are volatile fatty acids which can pro- vide most of the energy needed by the host animal. In addition, rumen microorganisms synthesize protein from non-protein nitrogen compounds, vitamin K and all of the B-complex vitamins. The protein is synthesized in the form of the microbial body and is transported to the small intestine where it is utilized in the same manner as in monogastric animals. Less than 10 per cent (1.0 billion hectare) of the total world land surface is planted to crops that produce food for human consumption. There is an ad- ditional 19 per cent (2.6 billion hectare) in permanent pastures and meadows, but it is improbable that all of this could be converted to crops edible by man. The ,world's forest lands yield approximately two-thirds of the total carbohydrate produced each year on the land surface. The remaining one-third comes from all other types of land plants; the majority of which is cellu- lose which cannot be utilized by man. Much of the cellu- lose now produced is poorly utilized even by ruminants primarily because of its high lignin content. However, methods are being developed for hydrolyzing the ligno- cellulose bonds and making the cellulose more available to the rumen microorganisms. It seems reasonable that, in time, these methods will be improved and/or new methods devised that will make it practical to prepare these feeds for ruminant consumption. In the harvesting of cereal grain and legume seeds, the stalks and leaves are left in the field in spite of the fact that, in some cases such as with corn, they con- tain up to 50 per cent of the total plant gross energy. Residues from other crops such as tubers could be converted by ruminants into high quality foods which would otherwise not be available to man. Another potential source of feed for ruminants are by-products of the food processing industries. Many of these energy sources are not only being wasted, but also pollute the environment. These energy sources are mostly deficient in protein and contain only low levels of the B-complex vitamins. Supplementation with non-protein nitrogen and feeding them to ruminants would result in their conversion to nutritious food for human consumption that has a high concentration of protein and B-complex vitamins. Non- protein nitrogen compounds can also be used by ruminants for improving the efficiency of conversion of plant pro- tein to food for man. The widespread use of non-protein nitrogen com- pounds in ruminant nutrition is relatively recent and has been primarily restricted to urea. Urea can make up approximately 50 per cent of the protein required by beef cattle and feeders use it to reduce feed cost. Poorer performance and toxicity frequently result when urea exceeds 50 per cent of the total crude protein intake. Therefore, there has been a search for non-protein nitro- gen compounds that can safely and economically meet a larger proportion of the protein requirements of rumi- nants. Extensive research conducted at the Michigan station with non-protein nitrogen addition to corn silage established that much of this nitrogen was combined with organic acids that resulted from fermentation to form ammonium salts. The performance of cattle fed non- protein nitrogen treated silages compared to untreated silages supplemented with soybean meal indicated a high availability of ammonium salts for feedlot cattle. In addition, it was hypothesized that these compounds could be produced by ammoniating organic acids produced from the fermentation of substances now considered waste pro- ducts. Therefore, the objectives of this study were to: (1) Compare ammonium salts of organic acids, soybean meal, and urea as sources of supplemental curde protein for feedlot cattle. (2) Determine the effect on nitrogen metabolism by adding organic acids to feedlot rations supplemented with urea and soybean meal. 5 (3) Describe the rumen and blood parameters associ- ated with the digestion and metabolism of ammonium salts of organic acids. (4) Determine the effect of high versus moderate levels of concentrate on the utilization of ammonium salts of organic acids. (5) Assess the potential of ammonium salts as nitrogen supplements and justification of future studies on methods of commercial production of ammonium salts of organic acids. LITERATURE REVIEW Historical Development of Protein Nutrition Protein is the most abundant nitrogenous compound in the diet and in the body. Consequently, the early history of protein metabolism is linked to the discovery of nitrogen and its distribution in nature. Munro (1964) and Stangel (1967) have reviewed early protein biochemistry. Daniel Rutherford is generally attributed with the discovery of nitrogen gas in 1772 but the name nitro- gen was not given to the new material until 1790. The French chemist Antoine Lavoisier reported in 1790 that nitrogen gas played no part in the metabolism of the mammalian organism. A system of organic analysis for nitrogen by treating the organic material with potassium chlorate was devised by Gay-Lussac in 1810. In addition, this period also saw the isolation of several compounds of considerable interest in the later study of protein metabolism. Urea was identified in the urine by H. M. Rouelle in 1773. In 1823, Pre'vost and Dumas demonstrated that urea accumulated in the blood when the kidneys were removed and led to their suggestion that the liver was the site of urea formation. By 1824, Proust made the first accurate analysis of urea and determined its em- pirical formula. In 1828, Wohler demonstrated the synthe- sis of urea from inorganic substances. Amino acids were isolated in 1810 by Wollaston and acid hydrolysis was first employed for disintegration of proteins by Braconnot in 1820. Advances in the knowledge of protein metabolism from the late 1820's until the turn of the century were hampered by inadequate analytical techniques and contro- versy over the relationship of dietary compounds to those found in the body. The major contributions included the recognition of protein as a component of animal and plant tissues (Muder, 1838 as reported by Stangel, 1967) and the development of nitrogen balance as a technique for the study of protein metabolism by Voit. In 1900, a partial quantitative analysis of indi- vidual isolated proteins was made possible by the method of Kossel and Kutcher who observed wide divergence in the amino acid content of different proteins. The es- tablishment of a link between defects in the nutritive value of a protein and its amino acid composition was made by Willcock and Hopkins in 1906 (Munro, 1964). Rose (1938) replaced dietary protein with mixtures of purified amino acids and culminated his study with the classifi- cation of amino acids as essential or non-essential for both the rat and man. This preceded studies by Osborne gE_§l. (1919), Mitchell (1923-24), Block and Mitchell (1946), Oser (1951), Miller and Bender (1955), and Bender and Doell (1957) that devised methods of evaluating pro- tein quality for non-ruminants. These methods involved the utilization of proteins by growing animals or by protein-depleted animals. Chemical methods for determi- nation of the amino acid content of the protein were also developed. Nitrogen Metabolism in the Ruminant Nitrogen metabolism in the ruminant has been the subject of several reviews (Blackburn, 1965; Hungate, 1966; Waldo, 1968; Chalupa, 1968; Conrad and Hibbs, 1968; Smith, 1969; Kay, 1969; McDonald, 1968 and Purser, 1970a, b). Microbes in the rumen degrade a major portion of the dietary and endogenous nitrogenous compounds. Some of the degradation products may be absorbed directly from the rumen or from the lower alimentary tract. However, most are used in the synthesis of microbial protein. The nitrogenous substances presented to the abomasum and in- testine of the ruminant consist of those present in the microorganisms and, to a limited extent, those that escape degradation and absorption in the rumen (Smith, 1969 and Chalupa 23.21'1 1972). Hutton §E_31. (1971) estimated that 50 per cent of the total nitrogen leaving the abomasum was bacterial nitrogen. Jackson gt_31. (1971) noted that about half of the nitrogen that passed to the duodenum was a-amino nitrogen. In addition, Black (1971) suggested that the complete degradation of dietary protein in the rumen would result in about 50 per cent less absorbed protein from the small intestine than in a non-ruminating lamb. Most of the nitrogenous material ingested by rumi- nants receiving natural feeds consist of proteins. These are usually broken down by the rumen bacteria to yield amino acids and then ammonia (Blackburn, 1965 and Hungate, 1966). Many individual species of rumen bacteria use ammonia as a nitrogen source in preference to amino acids and some species have an absolute requirement for ammonia (Smith, 1969). In contrast, the protozoa in the rumen are mainly ciliates (Hungate, 1966) and ciliates in general have an absolute requirement for amino acids (Kidder, 1967, as reported by Smith, 1969). 10 Al-Rabbat g£;213 (1971a, b) reported a procedure for estimating rumen microbial synthesis from ammonia. It was estimated that 61 per cent of the microbial nitrogen was derived from ammonia (Al-Rabbat gt_31., 1971a) and that the formation of microbial cells from ammonia was dependent on energy intake and independent of nitrogen intake (Al-Rabbat gt_31., 1971b). In this study they estimated that between 42 and 100 per cent of the total microbial nitrogen could be derived from ammonia. Beever gE_gl. (1971) fed low-nitrogen rye grass hay to sheep and also concluded that available energy rather than nitrogen limited microbial protein synthesis. Mathison and Milligan (1971) found that 50 to 65 per cent of the bacterial nitrogen and 31 to 55 per cent of the protozoal nitrogen were derived from ammonia. Diets consumed by ruminants normally contain appreciable nitrogenous material other than proteins. Pasture plants contain about 20-30 per cent of their total nitrogen as non-protein nitrogen or NPN (Ferguson and Terry, 1954 and Hogan, 1964) and corn silage contains a much greater proportion (Henderson §E_§1., 1971a). Most of the compounds in the NPN fraction are rapidly degraded in the rumen and form ammonia (Smith, 1969). Apart from NPN derived from natural feeds, ammonia compounds or other nitrogenous compounds such as urea are added to ruminant rations. 11 Post-Ruminal Metabolism of Protein The digestion and absorption of protein in the small intestine of the ruminant is very similar to that found in non-ruminants and has been reviewed by (Gitler, 1964). The hydrogen ion concentration in the abomasum permits the autocatylytic conversion of pepsinogen to pepsin. Rennin, the milk coagulating enzyme, is also found in the abomasum of the young calf. The object of proteolysis in the stomach appears to be one of splitting certain bonds in preparation for further hydrolysis by that proteases of the small intestine. It is also proba- ble that secondary linkages in proteins are broken by the acidity of the abomasum and result in denaturation (Gitler, 1964). The presence of acid, peptides and fats in the pyloric area of the abomasum and the duodenum induces the release of secretin and pancreozymin from the duodenal mucosa which stimulates secretion by the pancreas and by the Brunner's glands (Harper, 1959). The combined action of the proteolytic enzymes of the pancreatic juice and intestinal mucosa result in further digestion of exogenous nitrogen from the stomach (Geiger, 1951; Chen §E_gl., 1962). The absorption of the greast part of the protein 12 from the stomach occurs in the distal duodenum and proximal half of the jejunum (Schlussle, 1959). The main end-products of protein digestion that are absorbed across the mucosal membrane are amino acids (Gitler, 1964). Whole proteins are absorbed in the very young ruminant as a means of establishing passive immunity from disease (McCance and Widdowson, 1964). There is disagreement concerning the absorption of small peptides across the mucosal membrane. Amino Acid Supply and Requirements of Ruminants There appears to be no significant discrepancy between ruminants and non-ruminant mammals in the es- sential chemical pathways of nitrogen metabolism in the various tissues of the body (McDonald, 1968 and Hogan and Weston, 1970). Black §E_§1. (1952, 1957) and Downes (1961) demonstrated by using isotope techniques that sheep and cattle are not able to synthesize essential amino acids (EAA) at the tissue level. This has also been shown for man, rats and dogs. Rose (1938) made it clear that EAA are indispensable for maintenance and optimum growth. They must be provided in the diet or at the ab- sorption sites for ruminants in adequate amounts to meet requirements. 13 Rumen bacteria and protozoa influence the amino acid supply to the animal by degrading dietary protein and by synthesizing microbial proteins (Bryant and Robinson, 1961, Hungate, 1966 and Allison, 1969). The quantitative supply of amino acids is also influenced by nitrogen intake (Clarke, gE_al., 1966 and Williams §E_31,, 1953), nitrogen source in the diet (Little and Mitchell, 1967) and the microbial population in the rumen (Bergen and Purser, 1968 and Bergen gE_al., 1968). After absorption amino acids are transported to the liver where they are used for protein synthesis and gluconeogenesis. Some may also be transferred to the circulating plasma pool (Purser, 1970a). The use of amino acids for gluconeogenesis in sheep was estimated to account for about one—fourth of the total plasma glucose (Reilly and Ford, 1971 and Wolff §E_al., 1971). Several workers have interpreted an increase in plasma amino acid concen- tration as being indicative of improved protein status, but Zimmerman and Scott (1967) showed that an improved balance of amino acid led to a decrease in plasma amino acid concentration. There has been considerable research conducted to describe precisely the amino acid supply to the ruminant for metabolism but very little has been done to determine the adequacy of the supply (Purser, 1970a). Bergen (1967) examined the effect of ration on the protein quality of 14 rumen microorganisms. The data showed that rations did not significantly effect the amino acid composition of the rumen bacteria and rumen protozoa. Bergen §£_21, (1968) concluded that histidine and cystine were the limiting amino acids of rumen protozoal and bacterial pro- tines after feeding these proteins to rats as the only nitrogen source. Leucine, arginine and lysine were also in short supply. Leibholz (1971) used in vitro techniques to determine that L-histidine and glycine were transferred across the rumen epithelium in sheep. Methionine may be a limiting amino acid for ruminants (Nimrick §E_§1., 1971 and Wright, 1971). It is suggested that the hydroxy analog of methionine may es- cape rumen degradation and increase the amount of metnio- nine available to the animal. Results to date have been variable. Methionine hydroxy analog (MHA) has been shown to increase cellulose digestion in the rumen (Salsbury gE_al., 1964, 1967, 1970) and increase nitrogen retention (Polan et_31., 1970 and Salas, 1971). There have been reports of increased daily gain of steers fed MHA (Salas ‘gg_gl., 1971 and Burroughs §E_31., 1969b, 1969c) as well as reports in which MHA had no effect on daily gain of beef steers (Gosset gE_al., 1962; Beeson §E_al., 1970; Hale 2E_31., 1970a, 1970b; and Lofgreen, 1970). Other workers have reported a benefit (Ternus ep_al., 1971) or no benefit (Peter §E_§l.,; Ternus gE_al., 1971 and Wilson et al., 1971) from feeding MHA to sheep. 15 In several studies MHA was fed to dairy cows and equally conflicting results were obtained. Griel gt_al. (1968), McCarthy §E_§1. (1969), Polan gE_§1. (1970) and Bishop (1971) reported increased milk production with supplemental MHA. In contrast, there have been several reports that MHA had no effect on milk production (Williams gE_§1., 1970; Grugaugh and Olson, 1971; Kim gE_gl., 1971 and Hutjens and Schultz, 1971). Rosser gE_al. (1971) suggested that MHA may enhance triglyceride transport into the mammary gland. Both increased milk fat pro- duction (BishOp, 1971 and Kim etpalfl 1971) and no improve- ment in milk fat production (Hutjens and Schultz, 1971 and Grubaugh and Olson, 1971) have resulted with MHA supple- mentation. On the average, it appears that the feeding of MHA was not beneficial. However, there may be special circumstances that result in an increased need for methionine in ruminants. Urea as a Nitrogen Source in Ruminants The recognition that microorganisms in the rumen play a unique role in the nutrition of the host animal was reported independently by Zunte and Hageman in 1891 (reported by Stangel, 1967). Hageman pointed out that the confusion in experimental results found when testing the nutrative value of NPN compounds could be explained by the role that microorganisms play in the digestive system of 16 herbivorous animals. For the next 35 years, the ability of rumen organisms to utilize NPN was explored. Morgan gt_gl., in a series of experiments from 1907 to 1924, showed that 30 to 40 per cent of the protein in sheep rations could be replaced by urea (reported by Stangel, 1967). The first work in the United States on urea uti- lization was not conducted until the 1930's (Chalupa, 1968; Beeson, 1969 and Church gE_al., 1970). Hart §£_31. (1938, 1939) concluded that ruminants could use simple nitrogen compounds through the action of rumen micro- organisms. They also reported diuresis, increased blood urea and some kidney damage in animals on rations with over 60 per cent of the protein equivalent as urea. Bartlett and Cotton (1938) showed that dairy heifers could use urea effectively. The substitution of urea in the rations of producing dairy cows was re- ported by Owen (1941) and Owen 22.21: (1943). The supple- mentation of urea was found to have no effect on the percentage of protein, fat, lactose or total milk solids of the milk. The amount of urea in the milk never exceeded that of the blood. Loosli gtpglg (1949) obtained substantial growth over a three-month period with lambs and goats fed a purified diet devoid of amino acids in which all of the nitrogen was provided by urea. In the same experiment, EAA were 9 to 20 times greater in the rumen and in the 17 feces than in the feeds fed. Loosli 23.21: concluded that all EAA were synthesized by rumen microorganisms and this was later confirmed by Duncan gE_31. (1953). Other feeding trials with cattle (Rupel gt_§1., 1943; Willet 9131., 1946; Briggs 2131., 1947; Dinning gt_al., 1949 and Brown e5_al., 1956) have conclusively demonstrated the usefulness of urea for partially meeting the protein needs of ruminants. Nitrogen balance ex- periments (Harris and Mitchell, 1941; Johnson §E_gl., 1942; Harris et_21,, 1943; Hamilton ep_al., 1948 and Arias gE_§l., 1951) have added further information, showing that limited amounts of urea can be converted into protein. Urea is particularly suited as a feed ingredient since it is an economical, odorless material of high nitrogen content and biological availability (Belasco, 1954). Numerous studies at the Michigan Station (Henderson 2E_31., 1960; 1968a , b; 1970) have shown that urea can not only replace part of the supplemental protein but also substantially reduce the total feed cost for feedlot steers. Other workers (Ewing and Burroughs, 1963 and Martin §E_El°r 1968) have shown that urea can also be used in the rations of breeding cattle to reduce feed costs without sacrificing optimal performance. In contrast, experiments in Oklahoma have shown that urea is not as efficiently utilized as natural pro- teins for wintering cattle on native grass. This was true 18 for both steers (Nelson gE_al., 1961) and for lactating cows (Miller g5_al., 1958 and Williams §E_al., 1968). Williams §E_al. (1968) thought that the reason that pre- vious Oklahoma work had failed to show good urea utilization was due to the low level of energy in the ration. Allen (1972) found that a liquid protein supplement that derived 20 per cent of its crude protein equivalent from natural protein and 80 per cent from urea was more efficient than a supplement that derived all of its protein equivalent from urea. This was true for both yearling steers, and for calves nursing cows that grazed frosted winter pas- tures in Argentina. Newland EE_E£° (1961) reported a slight depression in gains and feed efficiency when urea made up 100 per cent of the supplemental nitrogen for cattle. In order to make the most efficient use of urea as a nitrogen supplement to poor quality roughages, a readily available source of carbohydrate appears necessary (McKnaught and Smith, 1947; Bell eE_al., 1953; Reid, 1953 and Belasco, 1956). Arias gE_§1. (1951) reported that increasing the energy content of the fermentation mixture resulted in an increased urea utilization with all sources of energy tested. This was true regardless of whether the energy source was a soluble carbohydrate such as dextrose or sucrose, or whether the carbohydrate was more complex such as the cellulose of a high fiber feed. 19 Potter §E_al. (1971) compared quantities of amino acids reaching the abomasum and plasma amino acids of steers fed different sources of protein. Only 79.4 per cent as much nitrogen reached the abomasum for steers fed urea compared to steers fed soybean meal. When 2.5 per cent molasses was added to the urea ration, the amount of nitro- gen reaching the abomasum was 92.5 per cent of that when soybean meal was fed. Commercial liquid supplements of molasses, urea, phosphorus, trace minerals and vitamins were first intro- duced to the United States in 1951 (Beeson and Perry, 1970). Feedlot tests with beef steers (Perry 2E_31,, 1967b; Gay and Vetter, 1967 and Kercher and Paulus, 1967) have shown no significant difference in the nutrative value or cattle response to high-urea dry or liquid supplements provided the supplements and/or rations contain the same essential nutrients in the proper balance. Workers at Purdue (Beeson gp_al., 1964 a, b, 1968; Beeson and Perry, 1969, 1970; Perry and Beeson, 1968 and Perry §E_§1., 1969) reported the existence of unidentified urea protein factors (UPF) in cattle fed high urea rations. The addition of both dehydrated alfalfa meal and dis- tillers grain solubles increased nitrogen retention, feed efficiency and average daily gain with urea supplements. Perry §E_§1. (1969) found a slight but non-significant increased daily gain by adding fish solubles to a high 20 urea liquid supplement. Burroughs §E_313 (1969a) also reported improved performance when fish solubles were added to high urea liquid supplements, but found no advantage to adding fish solubles to high urea dry supplements. Burroughs gE_31. (1972) reported that converting the nitrogen in the ration to crude protein equivalent using the 6.25 multiplication factor for the nitrogen present in all feed stuffs, irrespective of the type of nitrogen present, may at times lead to inaccurate protein evaluation when applied to NPN in the diet. They cited attempts to measure quantitative protein requirements of feedlot cattle that resulted in widely divergent values. Burroughs gE_31. (1971a) proposed a new system for defining protein requirements and the value of various cattle feed ingredients. The measurements employed were designated as metabolizable amino acids or metabolizable protein and were defined as the quantity of protein di- gested or amino acids absorbed in the post-ruminant portion of the digestive tract of ruminants. The amount of meta- bolizable protein required is calculated as the amount of protein required for maintenance plus the amount of pro- tein deposited during growth times 1.667. The factor 1.667 is used to account for the 40 per cent loss of amino acids during metabolism. 21 A measure of the amount of urea that can be useful in any given cattle ration was a second part of the meta- bolizable protein system( Burroughs 33431., 1971b). The urea fermentation potential (UFP) was defined as the esti- mated grams of urea per kilogram of feed dry matter con- sumed that is useful in fermentation by microorganisms in the fore part of the digestive tract of ruminants. The basis for establishing UFP values involves the amount of fermentable energy present in the feed and the amount of ammonia formed from protein degradation due to rumen fermentation of that feed. Burroughs g£_al. (1972) cited several instances in which the metabolizable protein system predicted accurately the cattle performance while the 6.25 multiplicative system did not make an accurate prediction. Urea as an Additive to Corn Silage Urea added to corn silage is hydrolyzed by urease contained in the fresh chopped corn plant resulting in higher levels of ammonia than untreated corn silage (Hastings, 1944 and Karr §E_31., 1965). The latter workers reported that 28 to 50 per cent of the urea added to corn silage at ensiling was hydrolyzed. Most of the hydrolyzed urea is recovered in the form of ammonia (Huber et_gl., 1968 b; Henderson, Purser and Geasler, 1970; and Lopez et al., 1970). Urea recovery from treated silage has 22 varied from 4 per cent to 84 per cent (Bentley eE_§1., 1955; Hatfield gE_31., 1966 and Huber et_§1., 1968b); how- ever, it is generally agreed that about 50 per cent of the urea applied remains as urea for silage in the dry matter range of 28 to 40 per cent. Urea treated silage was found to have a slightly higher pH than untreated silage (Davis gg_al., 1944 and Cullison, 1944). Klosterman gtpal, (1963) reported that the buffering capacity of ammonia arising from urea hydrolysis produced a higher pH and increased levels of lactate and acetate in urea treated silage. Formation of ammonium salts resulting from the combination of ammonia and organic acids produced in the silage have been sug- gested by Bentley §E_gl. (1955), Henderson §E_§l. (1971a), Henderson and Geasler (1969), Henderson, Purser and Geasler (1970) and Johnson et_al. (1967). A comparative feeding trial with urea treated corn silage and untreated corn silage utilizing lambs indicated that apparent drymatter digestibility was similar and crude protein digestibility was improved (Bentley gt_gl., 1955). Karr gt_§1. (1965) reported that lambs retain more nitrogen when urea was added at time of ensiling and Hatfield §E_31, (1966) concluded that supplemental energy was more effectively utilized when urea was added at ensiling. Henderson and Purser (1968b) reported dry matter consumption of beef heifer calves was less when 23 urea was added to the silage at time of feeding when com— pared to adding urea at ensiling and control silage supplemented with soybean meal. The latter two were simi- lar. Differences in gain were small for heifers receiving urea treated and urea supplemented silages and both urea groups gained slower than those fed control silage supplemented with soybean meal. Early work with urea treated silage showed reduced ration acceptability but no reduction in milk production (WOodward and Sheppard, 1944 and Wise §E_31., 1944). Huber §E_al. (1968a) found no difference in average pro- duction of cows fed urea treated silage or untreated silage. They suggest that high heat of fermentation may render nitrogen unavailable since the persistence of lactation was lower for cows fed high dry matter (44-45 per cent) corn silage treated with urea. When corn silage was treated with 0.5 per cent urea, a reduction in the natural protein in the concen- trate from 18 to 13 per cent did not depress milk yield, but yields were decreased on the lower protein concentrate without the urea in the silage (Huber §E_31., 1967). Huber gt_31. (1967) also reported that it may be possible to increase the level of urea in silage to 0.85 per cent when urea is not present in the concentrate. Owens §E_al. (1968) reported that milk yields were depressed when the ration contained 0.5 per cent urea in the silage and a l per cent urea grain mixture. 24 Urea Utilization, Ammonia Absorption and Urea Toxicity Helmer and Bartley (1971) cite many references that indicate that urea is rapidly hydrolyzed to ammonia and carbon dioxide in the rumen. However, Farlin gt_21, (1968a) questioned the theory that all urea is converted to ammonia before utilization. They observed that the carbon of urea did not equilibrate with the carbon dioxide pool, and suggested that urea is metabolized in the rumen without complete hydrolysis to carbon dioxide and ammonia, and that it need not be hydrolyzed for utilization of its nitrogen. A major problem in efficient utilization of urea is rapid release of ammonia. Bloomfield §E_al. (1960) indicated that urea hydrolysis uccurred four times faster than uptake of liberated ammonia by the microorganisms. Ammonia absorption is one way nitrogen disappears from the rumen (McDonald, 1948) and this apparently depends on the concentration gradient of ammonia and the pH of the rumen (Hogan, 1961). Some ammonia may be utilized by the rumen mucosa in synthesizing l-glutamate (McLaren §£_21., 1961 and Hoshino gE_31., 1966). It is well established that the consumption of large quantities of dietary urea in a short time can be toxic. Urea toxicity is more likely when urea is given as a drench (Dinning et al., 1948 and Word et al., 1969), 25 particularly if the diet is deficient in digestible carbo- hydrate (Dinning eE_gl., 1948) or if the animals have been starved or fasted (Clark et_§l., 1951). Lewis (1960) noted a direct relationship among rumen ammonia concen- tration, blood ammonia concentration and rumen pH. Coombe gE_gl. (1960) suggested that a high rumen ammonia concen- tration would not necessarily be toxic unless the pH rose above 7.3. This may be true with urea but not necessarily with other NPN compounds like ammonium acetate. Ammonium acetate has been reported to elevate rumen and blood ammonia concentration and produce toxicity without raising rumen pH (Webb and Bartley, as reported by Helmer and Bartley, 1971). Most performance studies with animals fed urea have indicated that the utilization of urea nitrogen is inferior to that of conventional protein supplements (Helmer and Bartley, 1971) especially for growing type rations consisting primarily of roughages (Perry gE_§l., 1967a) and for high urea rations for high producing dairy cows (Helmer and Bartley, 1971). In recent years, many NPN compounds other than urea have been explored in the hope of finding one that can safely, efficiently and economically meet a greater portion of the supplemental nitrogen requirements for ruminants than is feasible with urea as it is presently used. 26 Other Noneprotein Nitrogen Compounds Belasco (1954) compared various nitrogen compounds with urea, using artificial rumen techniques, cellulose digestion and ammonia concentration as criteria for evalu- ation. All 12 of the urea derivatives tested inhibited bacterial growth and were not extensively hydrolyzed. Amides of monocarboxylic acids (such as formamide, ace- tamide, etc.) were hydrolyzed to produce low levels of ammonia and good bacterial growth, but the resulting cellulose digestion was below that obtained with urea. The diamides tested were insoluble in water and, like the urea derivatives, were not hydrolyzable. Guanidine hydrochloride, guanidine acetate, and guanidine carbonate produce low levels of ammonia while supporting good bacterial growth and cellulose digestion. All of the other amidines tested resulted in inferior cellulose digestion and bacterial growth. Brent gE_§l. (1966) compared in vitro the microbial metabolism of urea, 1, 3-dimethy1-urea, biuret, biurea, guanidine hydrochloride, guanyl urea sulfate, and thio- carbanalide. Only urea appeared to be hydrolyzed to a useful degree. Accord gE_al. (1966) also used in vitro procedures to test the effectiveness of selected ammonium salts, amino acids, amides and amidines as nitrogen sources for starch digestion by rumen microorganisms. Aspartic acid was inferior to urea but significantly more effective 27 than the other amino acids tested. Acetamide, propionamide, butyramide, succinimide, malonamide, guanadine acetate, or amino guanadine bicarbonate did not affect starch di- gestion consistently. Simpson and Jones (1967) contra- dicted Belasco (1954) and Brent gE_al. (1966) when they indicated that urea derivatives can be hydrolyzed and will support in vitro cellulose digestion. Crystalline urea, methyl urea and glycourea promoted the greatest increase in cellulose digestion. Repp gt_§l. (1955b) determined the toxicity and comparative feeding value of several NPN compounds. The amides (propionamide, formamide and biuret) were nontoxic, while all ammonium salts of organic acids except ammonium succinate were toxic when administered orally in large doses. Repp gE_al. (1955a) reported that urea, formamide or prOpionamide appeared to support growth of lambs when they replaced 50 per cent of the protein nitrogen of the ration, but formamide was inferior. When less than 30 per cent of the protein nitrogen was replaced with pro- pionamide or urea, the gains were equal to those obtained with natural protein. Growth data suggested that lambs require two to four weeks to become fully acclimated to the NPN compounds tested. Of all the NPN compounds considered as substitutes for urea, biuret probably has received the most attention. But many early investigations were rather unproductive 28 because researchers were working with biuret that con- tained up to 50 per cent urea and most were unaware that biuret requires a long adaption period (Helmer and Bartley, 1971). Nitrogen balance studies as well as production studies have indicated that biuret is utilized somewhat better by sheep than by cattle. Hatfield eE_§1. (1959) and Campbell gE_al. (1963) indicated that nitrogen reten- tion for cattle was greater for urea than biuret, but Gaither gt_al. (1955) and Tomlin §E_31. (1967) reported greater nitrogen utilization for biuret than for urea for lambs. Growth studies with sheep (Meiske §E_31,, 1955 and Hatfield gt_al., 1959) resulted in equal perfor- mance for biuret and urea while similar studies with cattle resulted in inferior performance with biuret when com- pared to urea (Campbell §E_a1., 1963). Bucholtz and Henderson (1971) reported a greater average daily gain for biuret fed steers than for those fed urea or starea (starch coated urea compound). Most researchers have confirmed that utilization of biuret by ruminants becomes more efficient with time. Apparently, efficient utilization of biuret requires a longer acclimation period than urea (Oltjen gE_31., 1969) and an animal adapted to urea is not necessarily adapted to biuret (Johnson and McClure, 1967; Farlin gg_al, 1968b and Oltjen et al., 1968). The length of time required 29 for adjustment to biuret is disputed and ranges from three to five weeks (Welch §E_§1., 1957; Campbell eE_al., 1963; Tomlin gt;21,, 1967 and Oltjen 2E_al., 1969). Another NPN compound which has been considered as a substitute for urea is diammonium phosphate (DAP). It is generally agreed that DAP is less likely to cause toxicity in ruminants than urea, but agreement is lacking on its relative value, as indicated by nitrogen retention and animal performance (Helmer and BArtley, 1971). Palatability seems to be a primary problem associated with DAP (Lassiter §E_§1., 1962 and Oltjen e£_al., 1963). Dicyanodiamide has been evaluated as a protein or urea substitute. There have been reports of loss of weight by lactating cows (Rust ep_§l., 1956) or decreased milk production (Davis gE_§l., 1956) or decreased palatability of the ration (Lassiter gE_al., 1955) from feeding this compound. However, Lassiter gg_al. (1955) and Rust §£_al. (1956) reported no significant differences in milk pro- duction between urea and dicyanodiamide supplemented cows. Magruder and Knodt (1954) obtained similar weight gains in dairy heifers fed a basal ration supplemented with either soybean meal, urea or dicyanodiamide. Included in numerous NPN sources investigated are ammoniated cane molasses and ammoniated products of the milling industry. In vitro studies (Stallcup, 1954; Davis et al., 1955 and Hershberger et al., 1955) indicated that 30 only the free ammonia of ammoniated products was utilized by rumen microorganisms. Hughes et;gl. (1955) reported similar digestion coefficients for rations containing either soybean meal or ammoniated cane molasses, but other research reviewed indicated a marked depression in the digestibility of ammoniated cane molasses (Tillman gE_al., 1955; 1957b) as well as in ammoniated industrial products (Tillman and Swift, 1953 and Tillman gt_213, 1957a). Frye gt_gl. (1954) and Parham gE_31. (1955) reported growth studies with dairy heifers in which similar gains were made by heifers receiving either cottonseed meal, urea or ammoniated molasses as the supplementary nitrogen source. In contrast, poor results were obtained when ammoniated molasses was used as a protein source in winter rations for beef calves (Richardson gE_al., 1954, as reported by Helmer and Bartley, 1971) or in fattening rations for cattle (Tillman gE_gl. 1957a) or lambs (Tillman et al., 1957b). Pro-Sil Addition to Corn Silage A suspension of ammonia, minerals and molasses (Pro-Sil) has been developed by Michigan workers for ad- dition to corn silage at ensiling time. Pro-Sil was formu- lated to make corn silage a balanced ration for feedlot cattle (Henderson g£_31., 1970b), non-lactating dairy cattle or cows producing below 30 pounds of milk (Huber and Hillman, mimeo D-236). 31 Henderson §E_21. (1971c) reported that corn silage treated with Pro—Sil significantly (P < .01) increased total nitrogen, water insoluble nitrogen, non-protein nitrogen, ammonia nitrogen, lactic acid and pH when com- pared to untreated corn silage. Acetic acid content in Pro-Sil treated silage decreased significantly (P < .01). This is in agreement with the other work (Beattie eE_al., 1971). Beattie §£_gl. (1971) reported that nitrogen fractionization of the Pro-Sil treated silage revealed 21 per cent of the increase in total crude protein was in the form of water insoluble protein, 58 per cent in the form of ammonium salts, and 21 per cent remained as unidentified nitrogen compounds. Approximately 95 per cent of the nitrogen added as Pro-Sil was accounted for by the increased crude protien content of the Pro-Sil treated silage. Growth studies comparing Pro-Sil treated corn silage with urea treated corn silage and untreated corn silage supplemented with soybean meal have resulted in no significant difference in daily gains due to nitrogen sources (Beattie et_§1., 1971 and Henderson eE_al., 1971a, b, c, d). In addition, little difference in feed consumption was reported for the various protein sources and feed cost favored the NPN treated silages in all cases. Henderson et al. (1970a) reported that steers fed Pro-Sil treated 32 rye silage gained faster and more efficiently than steers receiving urea supplemented silage and feed costs were lower for the Pro-Sil fed steers. Higher milk yields, less average change per day and greater milk persistencies have been noted for high producing cows fed Pro-Sil treated silage than those fed control silage or silage treated with urea or urea plus minerals (Huber and Thomas, 1971; Huber gt_§l., 1971 and Huber and Hillman, mimeo D—236). Their data also indicate that Pro-Sil will maintain production even on silage of higher dry matter content (40 per cent ) which is an advantage over urea treated corn silage. Nitrogen balance studies with beef steers comparing silages treated with Pro-Sil, urea—minerals, or supple- mented with soybean meal revealed no difference in dry matter digestibility, nitrogen digestibility or nitrogen retention (Beattie, 1970). Dry matter digestibility and nitrogen utilization were not different in Pro-Sil or urea treated corn silages fed both lambs (Henderson 23.2%: 1970b) and lactating cows (Lichtenwaler et_21., 1971). Either urea or Pro—Sil addition to corn silage result in the formation of ammonium salts of the organic acids contained in the silage (Bentley 22.21:! 1955; Henderson 2E_21., 1969, 1970b, 1971a; Johnson §E_§l., 1967; and Beattie §E_§1., 1971). The literature cited indicates NPN treated silages will support performance similar to 33 soybean meal supplemented silages at a reduced cost. Therefore, interest in the utilization and production of ammonium salts has expanded in recent years. Ammonium Salts As noted previously, Belasco (1954) used in vitro techniques to compare various nitrogen compounds with urea. Each individual experiment had positive (urea) and negative (no nitrogen compound added) controls. The metabolic response of the various nitrogen compounds was expressed as a percent of the urea response. The experiments with ammonium salts of various organic and inorganic acids illustrated a high availa— bility of their nitrogen to the rumen microflora. In general, the main difference between the ammonium compounds and the amides and amidines was the high level of ammonium ion noted when introduced into the system. Belasco hypo- thesized that an excess of ammonium salts might not be as hazardous as a similar excess of urea because of the accompanying acid radical. The results of Belasco's work with ammonium salts are summarized in Tables 1 and 2. Except for ammonium nitrate, all ammonium salts tested (Table 1) provided nitrogen for rumen bacterial growth and subsequent cellulose digestion. Nitrogen utilization, Whatever a determination was analytically 34 possible, was comparable or superior to urea. Data could not be accurately obtained in systems containing ammonium sulfamate, nitrilotrisulfonate and triamidodiphosphate due to the instability of the materials when subject to analytical conditions. Ammonium nitrate proved toxic to the rumen microflora. Nitrogen utilization was greater for ammonium formate, succinate, lactate, a-ketoglutarate and malate than for urea (Table 1). Belasco suggested that the organic fragments of these compounds might enter into some biosynthetic process that stimulated nitrogen fixation by rumen microflora. Salts of other acids associated with the citric acid cycle (citrate, fumrate and pyruvate) were comparable to urea. Belasco further reported that ammonium succinate and lactate resulted in lower levels of free ammonia and a higher nitrogen utilization than urea at both 4 and 24 hours after introduction (Table 2). Ammonium formate showed the same trend, but to a lesser degree. The rapid utilization of ammonium succinate and lactate could not be duplicated by the addition of energy to urea as glucose in an amount equivalent to the lactate ion. Nor could this effect be fully duplicated by the addition of equivalent amounts of sodium succinate or succinic acid to urea. Belasco thought it was possible that the supple- mental succinate or succinic acid was metabolized before 35 m>aummms map 30Hmn mumu :0fiummmflp mmoHsHHmo m mmBMOHpcfl msHm> m>aummmc e .HOH ucoo N .Aemmav commamm someH msoz «man 0 messes: emz seem so In- mussemoseAeoeAsmAsu emz poow hm III SHMQOHHSmHuuoHHHuflc emz @000 mu III mumfimmasm vmz unmaamoxm hm Hm wumconumo emz usmaamoxm as as shamans emz unmaamoxm hm mm mumumom vmz Asmaamoxm as ea museses emz unmaamoxm em ooa mpmuuwo vmz Asmaamoxm OOH mos message“ emz ucmHHmoxm em moa mum>su>m vmz ucmaamoxm mm moa mumamfi wmz ucmHHmoxm eoa boa mmenom emz ucmaamoxm moH oaa mumumudamoumxno vmz ucmHHmoxm Hm «Ha mumuoma vmz unwaamoxm om mHH mHMCHOODm vmz ucmaamoxm ooH ooa men: nu3onw cowummmma :oaumeafluD pasomfiou Hmflumuomm mmoHsaamU smoouufiz mmcommmm mmHD mo w H .muamm EdwaoEEm on mmcommmn oaaonmumzll.a magma 36 Table 2. Comparison of selected ammonium salts.1 Ammonia Nitrogen Cellulose Level Utilization Digestion 2 mg after: % afterz. (%) Compound 4 hr 24 hr 4 hr 24 hr No nitrogen 22 9 0 0 25 Urea 225 106 25 77 79 NH4 formate 215 102 32 82 81 NH4 lactate 181 80 59 87 74 NH4 succinate 191 78 51 90 77 1From Belasco (1954). 2187 mg nitrogen was used for all but the no nitrogen negative control. all the ammonia was available from hydrolysis of urea and therefore could not affect the rate of nitrogen fixation. Wetterau and Holzchub (1960 and 1961, as reported by Coppock and Stone, 1968) reported that ammonia from ammonium salts is released more slowly than from urea in the rumen and this may facilitate rumen microbial growth. Accord EE_El° (1966) also used in vitro procedures to test effectiveness of selected ammonium salts, amino acids, amides and amidines as nitrogen sources for starch digestion by rumen microorganisms. Ammonium salts of sulfate, chloride, acetate and phosphate were equivalent to urea at all levels tested. They associated increased 37 ammonia levels after 4 or 8 hours of fermentation with rapid starch digestion. These results are in general agreement with Belasco (1954). Repp gE_§1. (1955a, b) determined the toxicity and comparative feeding value of several NPN compounds for lambs. When administered orally in large doses, all ammonium salts of organic acids except ammonium succinate were toxic (Repp gt_§l., 1955b). When urea, ammonium acetate, ammonium propionate and ammonium formate replaced as much as 50 per cent of the protein nitrogen in the ration, all NPN compounds appeared to support growth in lambs equally. At the 50 per cent level the NPN compounds promoted gains below those from protein sources. Moore and Anthony (1970) reported that ammonium acetate was more toxic and that ammonium lactate was less toxic than urea for sheep. Varner and Woods (1971) reported two metabolic studies with ammonium salts of organic acids, urea and soybean meal. In their first metabolic study they com- pared urea and soybean meal with two mixtures of ammonium salts. The two mixtures were referred to as a high ace- tate supplement and a high prOpionate supplement. They consisted on 75 per cent ammonium acetate, 15 per cent ammonium propionate, 10 per cent ammonium butyrate and 30 per cent acetate, 40 per cent ammonium propionate and 30 38 per cent ammonium butyrate, respectively. In the second metabolic study urea was compared with ammonium acetate, ammonium propionate and ammonium butyrate. There was no differences in digestibilities of dry matter, organic matter, cellulose or crude protein among the nitrogen supplements in either metabolic study. The total volatile fatty acid (VFA) concentrations of the rumen fluid samples was higher for the ammonium salts in both trials. Varner and Woods also reported that rumen pH was higher for urea in both trials and the change in rumen VFA for the ammonium salts reflected the individual ammonium salt or mixture fed. In the first metabolic study, rumen ammonia levels were higher at one hour post-feeding for steers fed ammonium salt mixtures than for urea. Serum urea levels were highest for urea next for ammonium salts and lowest for soybean meal treatments. Steers fed soybean meal also had lower rumen ammonia levels at all sampling times. In the second metabolic study, daily nitrogen retention was 6.6 g, 6.3 g, 3.8 g and 0.7 g for steers fed ammonium propionate, ammonium butyrate, ammonium acetate and urea, respectively. Steers fed ammonium acetate had the highest rumen ammonia levels. In contrast to the first study, serum urea levels were higher on ammonium acetate or ammonium butyrate than on ammonium propionate or urea . 39 Varner and Woods (1970) administered soybean flour, urea, ammonium acetate, ammonium propionate and ammonium butyrate at isonitrogenous levels through a stomach tube to portal cannulated lambs. The portal blood ammonia at one hour after feeding was 1055, 920, 650 and 425 micro- grams/m1 for lambs given urea, ammonium propionate, ammon- ium acetate, ammonium butyrate and soybean flour, res- pectively. Reductions in portal blood ammonia were noted when either ammonium acetate or urea plus acetic acid were compared with an isonitrogenous level of urea. Varner 23123: (1968) reported that a high propionate ammonium salt mixture was superior to urea and a high acetate ammonium salt mixture and almost equal to soybean meal in promoting gains in finishing trials for cattle. The high acetate mixture of ammonium salts was equivalent to urea. Varner and Woods (1969) reported that the gains of cattle fed ammonium salts of acetate, propionate or butyrate in a growing trial were significantly higher than for those fed urea. Utilization of Volatile Fatty Acids Virtually all the carbohydrate ingested by the ruminant is degraded by anaerobic microorganisms to vola- tile fatty acids (VFA) and ruminants are primarily dependent upon these acids (principally acetic, propionic and butyric) for their supply of energy (Johnson, 1955 and Carroll and 40 and Hungate, 1954). Carroll and Hungate (1954) estimated that the energy from VFA production in the rumen provided 70 per cent of the total energy required. Other workers (Balch, 1958 and Connolly E51233! 1964) have estimated that VFA account for 36 to 42 per cent of the digested energy. The gross kcal/g for VFA were reported as approxi- mately 3.5, 5.0 and 6.0 for acetate, propionate and butyrate, respectively (Emery, 1965). However, the total energy furnished by these three acids were reported as approximately equal (Carroll and Hungate, 1954). In addition, Orskov et_§1. (1969) found no differences in utilization of the energy from acetate and propionate, but there were differences in the partition of energy into milk or body tissues. More of the acetate was secreted in milk of lactating cows and more of the propionate was retained in body tissue. The VFA produced in the rumen are readily absorbed through the rumen wall (Barcroft gt_gl., 1944). The rumen epithelium is active in the conversion of n-butyrate to beta-hydroxy butyrate (Seto and Umezu, 1959 and Ramsey and Davis, 1965). Acetate and butyrate that escapes oxi- dation by the rumen epithelium are converted to ketone bodies by the liver; the process is inhibited by propionate (Seto et al., 1959 and Johnson, 1955). 41 There is disagreement concerning the relative rates of absorption of VFA from the rumen (Gray and Pilgrin, 1951). Sutherland (1963) reported absorption rates for acetate, propionate and butyrate were 1.6, 1.9 and 2.0 meq./hour per meq./liter in the rumen, respectively. Tsuda (1956) reported that there was no difference in absorption rates of free fatty acids in the rumen. How- ever, free fatty acids were absorbed faster from the rumen than the sodium salts of organic acid. The rate of absorption was greatest for sodium acetate and lowest for sodium propionate. Sodium butyrate was intermediate. Apparently, the absorption of free acetate is regulated by the concentration gradient between the blood and the rumen (Masson and Phillips, 1951 and Tsuda, 1956). The efficiency of utilization of VFA for different physiological functions has been studied by several meth- ods. Differences in the propotion of acetic, propionic and butyric acids in VFA mixtures continuously infused intra-ruminally had only a small effect on the efficiency of energy utilization in fasting lambs (Armstrong, Blaxter and Graham, 1957). For fattening, all acids were utilized less efficiently than for maintenance; this was particu- larly true for acetic acid (Armstrong gE_al., 1958). With salts of VFA, no difference could be found between acetate, propionate and butyrate in their ability to promote growth in lambs on an equal weight basis (Orskov and Allen, 1966a, 42 b, c and Orskov gt_§l., 1966). Armstrong and Blaxter (1957) stated that acid administration did not interfere with the normal process of rumen fermentation or impose non-physiological conditions upon the animals. Oxidation of the acids to provide the energy is not their only use by the animal. Studies with labelled acetic acid have shown that this acid is incorporated into many compounds. Lipogenesis in mammary tissure results in incorporation of acetic acid into milk fat (Folly and French, 1950 and Kleiber §£;31,, 1952). Acetate can also act as a precursor of the milk constituents lactose and glyceral (Popjack gp_§l., 1952) and the carbon moieties of non-essential amino acids in milk protein (Black §E_21., 1957). Acetic acid is also used in the synthesis of liver and body fat (Rittenberg and Bloch, 1945), the lipids of wool (Sjoberg, 1956) and cholesterol. Sheppard §E_al. (1959) reported that acetate was also used in the formation of propionic, butyric and higher acids in the rumen. They estimated that 12 per cent of acetate was converted to butyrate and 5 per cent to propionate. Propionate is a major precursor of glucose in the body (Armstrong and Blaxter, 1957 and Leng and Annison, 1963). Increasing the proportion of concentrates and pelleting increases in the proportions of propionate and butyrate produced in the rumen (Bauman et al., 1971; Davis 43 1967 and Hawkins gE_al,, 1963). Lactic acid administration also increases ruminal propionate or butyrate levels (Montgomery §E_al., 1963). Butyrate is involved in the synthesis of lactose (Kleiber gE_al., 1954) and can provide the carbon moieties of amino acids in milk protein (Black gE_al., 1952). Ramsey and Davis (1965) reported studies with labelled butyrate that indicated that n-butyrate was metabolized via the citric acid cycle in the rumen. The work of Elsden (1945), Phillipson (1952), Waldo and Schultz (1956) and Hueter et_al. (1956) indi- cates that lactate is converted to other fatty acids, especially propionate. Other workers report that the primary VFA resulting from lactate metabolism in the rumen was acetate (Jayasuria and Hungate, 1959 and Bruno and Moore, 1962). Bruno and Moore (1962) used uniformly labelled lactate and reported that 35 per cent of lactate was converted to acetate, 9 per cent to propionate, 33 per cent in ether extracted aqueous residues and the remainder in other fatty acids and fermentation gases. Hueter §E_§l, (1956) reported the infusion of sodium lactate in the rumen resulted in a large increase in blood lactate which was followed by an increase in blood glucose. Annison §£_213 (1963) reported that lactate is the precursor for 15 per cent of the glucose pool and that lactate accounts for 7 per cent of the CO2 production, 44 primarily via glucose. Salts of lactic acid (calcium, sodium and ammonium' were shown to alleviate ketosis (Seekles, 1951 and Shaw et al., 1955). The Effect of Organic Acid Additions on Intake Manning gt_al. (1959) suggested that blood acetate concentration in ruminants may be a chemostatic factor in controlling intake similar to glucose in non-ruminants. Acetic acid is the only fatty acid normally found in signi- ficant amounts in peripheral blood and its concentration changes with time after feeding (Balch and Campling, 1962). Intravenous infusion of sodium acetate, acetic acid and propionic acid depressed intake in cattle (Dowden and Jacobson, 1960). Intravenous infusions of glucose, butyrate, valerate, hexanoic acid and lactate did not affect intake. Holder (1963) noted that intravenous in- fusions of either glucose or acetate did not affect in- take in sheep. Intraruminal infusions of acetate have been shown to decrease intake (Simpkins §E_31., 1965; Weston, 1966; Rook gE_§l., 1963 and Baile and Pfander, 1965). Montgomery 2E_§1. (1963) observed a greater effect due to acetate than prOpionate or butyrate. The regulation of intake by VFA appears to be in the rumen since no depression was noted after abomasal infusions of acetate (Baile and Mayer, 1967) or duodenal 45 infusions of propionate (Egan and Moir, 1965). Acetate receptors are more likely to be located on the lumen side of the reticulorumen than in an area where they respond to blood acetate since intraruminal infusions of acetate depress intake and intravenous infusions do not (Baile and Mayer, 1968). These authors suggested that the feed intake depression following an acetate infusion is related to satiety. Many researchers have attempted to relate vola- tile fatty acids present in the silage to a reduction in consumption. Dinius gp_§l. (1968) added acetic acid to green chop and corn silage and reported that added acetic acid reduced dry matter intake but did not effect caloric intake. Wilkins 35121: (1971) reported a negative corre- lation between the acetic acid content of 70 grass and legume silages and consumption by sheep. Wilkins 22.21: also reported a positive correlation between voluntary intake of silage and silage lactic acid as a percentage of total acids. Hutchinson ep_al. (1971) reported that free acetic acid infused into the rumen reduced silage intake, but addition of the same quantity of acid to silage did not effect intake over a 24-hour period. Senel and Owen (1967) observed no reduction in voluntary intake when 2 per cent acetate, 1 per cent butyrate, or a combination of these acids were added to a 46 hay-concentrate ration. However, a mixture of 4 per cent acetate and 2 per cent butyrate appeared to cause nasal irritation and reduced intake. Later work by Senel and Owen (1966) using sorghum silage showed increased dry matter intake when acetate was added at levels up to 2.8 per cent of the ration dry matter. Lactate addition to sorghum silage at a low level (5.90 per cent DM) decreased intake but at the high level (9.03 per cent DM) intake was greater than the control silage. Acetate and lactate improved feed efficiency, when compared to the control. They concluded that something other than acetate and lactate depressed silage consumption. Emery §E_31. (1961) reported that lactic acid addition to corn silage reduced appetite in proportion to its concentration when fed to growing heifers. Feed efficiency increased in direct proportion to the lactic acid intake. There are conflicting opinions regarding the effect of lactic acid content of grass silage on dry matter consumption. McLeod et_al. (1970) reported reductions in dry matter intake were prOportional to the amount of lactic acid added and the resulting decrease in pH. Other researchers; King, 1943; and McCarrisk gE_gl., 1966) attribute the reduced intake of grass silages to the content of total organic acids. 47 The type of lactic acid used to study the effect of lactate on dry matter consumption must be evaluated. Dunlop and Hammond (1965) reported the L(+) isomer is more readily metabolized in the rumen and liver than the D(-) isomer. The lactic acid in corn silage consists of both the D and L forms; however, the D(-) isomer is more B " .41.“ abundant (Schaadt and Johnson, 1968). Summary As knowledge of protein chemistry and nitrogen metabolism of the ruminant animal evolves there is a greater emphasis on using NPN to replace natural protein in the diet. Extensive research with urea has established its advantages and limitations, but there is only limited information on other NPN sources. In vitro work with NPN compounds indicates that ammonium salts of organic acids are equal or superior to urea. Limited in vivo work indicates that ammonium salts may be utilized as a supplemental nitrogen source for ruminants. There have been no reports of comprehensive growth or metabolic studies with ammonium salts other than salts of acetate, propionate and butyrate. Other research has demonstrated that the organic acid portion of ammonium salts can be utilized for energy 48 either by the rumen microorganisms or by animal tissues. However, the addition of organic acids, especially acetic acid, to the ration may cause a reduction in dry matter intake. MATERIALS AND METHODS The studies involved in this dissertation include two feeding trials comparing various ammonium salts with more conventional nitrogen supplements, two feeding trials to study the effect of added lactic or acetic acid and three metabolic studies. Materials and methods are pre- sented under experimental headings. Experiment I--Feeding Trial Comparing Ammonium Acetate, Ammonium Lactate, Urea and Soybean Meal Design: Twenty-four cross bred yearling steers were randomly assigned by weight to one of the following treatments: soybean meal, urea, ammonium acetate or ammonium lactate. Harvesting of Feed: High moisture shelled corn and corn silage were harvested from a stand of hybrid corn averaging approximately 35 metric tons of 35 per cent dry matter (DM) silage or 5 metric tons of 85 per cent DM shelled corn per hectare. The corn silage received no additives and was harvested between September 1 and September 23, 1970. After harvesting at an average 36.7 per cent DM, the silage was stored in a 9.1 x 18.3 m. silo. The high moisture shelled corn Was harvested in October and placed in sealed storage. 49 50 Production of Ammonium Salts: Ammonium lactate and ammonium acetate were produced in 15 1. batches by neutralizing the respective acids with acqua ammonia until a pH of 6.5 was obtained. Technical grade 88 per cent lactic acid was used for the production of ammonium lactate and 8 1. glacial acetic acid was diluted with 7 1. of water for the production of ammonium acetate. This equal- ized the molar concentration of acid on the wet basis and provided for approximately 44.5 per cent crude protein solution (7.13 per cent N) for both ammonium salts. Each 15 1. batch was analyzed for nitrogen by the micro— Kjeldahl method to establish the amount of nitrogen fed. The average crude protein equivalent was 112 per cent and 81 per cent on a DM basis for ammonium acetate and ammonium lactate, respectively. Feed Analysis: Silage and shelled corn samples were taken every Monday, Wednesday and Friday during the feeding trial. Dry matter was determined on each sample for intake estimates by oven-drying the samples at 105° C. for 24 hours. Composite samples of moist silage or shelled corn were analyzed for nitrogen and organic acid fractions and then expressed on a DM basis from analysis of a paired sample dried at 55° C. for 48 hours. Total nitrogen was obtained by macro-Kjeldahl procedures. Soluble extracts of silage and shelled corn were prepared by homogenizing 25 g. of fresh sample with 100 m1. of deionized, distilled 51 water for one minute. The pH of the solution was deter- mined using a Corning Model 12 pH meter. The solution was strained through two layers of cheesecloth and total water soluble nitrogen was determined by micro-Kjeldahl. A second sample of extract was deproteinized with 1 m1. of 50 per cent sulfosalicylic acid (SSA) per 9 ml. -- -’ of extract, and a micro-Kjeldahl analysis of this fraction gave the water soluble non-protein nitrogen. The micro- diffusion method of Conway (1950) was used to determine the ammonia and urea fractions in the water soluble NPN fraction. The remaining extract was deproteinized with 1 ml. of 50 per cent SSA per 9 m1. extract as before, and then centrifuged for 10 minutes at 18,000 r.p.m. Volatile fatty acid content of the samples was determined by in- jecting samples of this into a Packard gas chromatograph, and lactic acid content was determined by the colorimetric method of Barker and Summerson (1941). The average chemical analysis of corn silage and high moisture corn fed during the trial are shown in Table 3. Feeding Trial: The yearling Hereford-Angus cross- bred steers utilized in this experiment originated in Texas and were purchased as fall calves on July 22, 1970, at an average weight of 253.8 kg. They were placed in outside lots and fed a full feed of corn silage until the smaller 52 Table 3.--Experiment I: Average chemical analysis of feeds fed.1 Corn High Moisture Observation Silage Sh. Corn Percent DM 34.51 74.16 Crude Protein Fractions: Total crude protein 8.99 9.46 Organic protein 4.68 7.83 NPN protein 4.31 1.63 ' Ammonium salts .65 .18 Urea .02 .00 Unidentified 3.64 1.45 Organic Acid Fractions: Total organic acid 9.44 .33 Lactic acid 7.75 .32 Acetic acid 1.67 .01 Butyric acid .02 .00 pH 3.89 5.46 1 Each value is the mean of 18 different composite samples taken every two weeks during the feeding trial. 53 steers were placed on this experiment November 3, 1970, at an average weight of 294.9 kg. The various nitrogen supplements were compared on a ration composed of 75 per cent concentrates (high moisture shelled corn, nitrogen supplement and mineral supplement) and 25 per cent untreated corn silage on a DM basis as shown in Table 4. The soybean meal and urea were fed as dry supplements combined with minerals and a dry mineral mix was formulated to be fed with the ammonium salts (Table 5). All ration ingredients were combined and mixed in a horizontal mixer prior to each feeding. The steers were implanted initially with 24 mg. of diethylstilbesterol (DES) and injected with 2,000,000 I.U. of vitamin A. Four months later, all steers were re-implanted with 36 mg. of DES and re-injected with 2,000,000 ‘units of vitamin A. The steers were individually weighed on two successive days at the beginning and end of the trial. The average of the two successive weights was used as initial and final weights. Cattle were group weighed every 28 days during the experiment. The feeding trial was terminated after 170 days on feed. The cattle were utilized in a metabolic study (to be discussed later) for nine days, then trucked 161 km., allowed to stand overnight, and slaughtered the next morning. After 48 hours in the cooler, carcasses were 54 Table 4.--Experiment I: Percent ration composition on a dry matter basis. Ingredient Lactate Acetate Soy Urea Corn silage 24.04 24.34 25.00 25.00 Shelled corn 65.74 66.54 68.36 68.36 Soy SUPP-l ---------- 6.64 ----- Urea supp.1 --------------- 6.64 Mineral supp.l 6.39 6.46 ---------- NH4 acetate2 ----- 2.66 ---------- NH4 lactate3 3.83 --------------- TOTAL 100.00 100.00 100.00 100.00 1See Table 5 for formulation. 2112 per cent crude protein (2.66 per cent of DM intake). 381 per cent crude protein (3.83 per cent of DM intake). ribbed, graded by a federal grader and fat and lean tracings were made of the 13th rib for determining cutability, fat thickness and ribeye area. The rib eye area was then determined by following the rib eye tracing with a plano- meter and the fat thickness perpendicular to the outside layer was measured three-fourths of the distance up the rib eye from the chine bone. The per cent kidney, heart and pelvic fat was estimated by the federal grader and the per cent boned, trimmed retail cuts was estimated using the USDA formula. 55 oo.ooH oo.ooH oo.ooa A¢BOB n>.mm mo.>h III: cuoo Umaamcm .HU III: NH.mH III: AZwva moms momma comm n--- u--- eo.om A.uogm wont Hams ammnsom mm.m nm.m mv.m inN refine “Ham Hmumcfis mouse mv.m oo.m no.m Amwm.mmv mHMMHSm Edapom mm.m nm.m mv.m imwm.maumowo~c mumnmmonm asHUHMOflo ucmfimammsm ucmEmHmmsm ucmEmHmmdm Ll ucmflwmumcH Hmumcwz Hmumcflzlmwno Hmnmcwzlmom w so wa mo ucmouomv mucmfimammsm mo .Amflmmn umuuma who cowumHSEHom "H ucwfiflummxmll.m magma 56 Experiment II--Feeding Trial Comparing Various Ammonium Salts of Organic Acids with More Conventional Nitrogen Sipplements Design: An 8 x 2 factorial design was utilized to study the following treatments: A. Two levels of concentrate (l) (2) 40 per cent of the ration DM as shelled corn and 60 per cent as untreated corn silage. 80 per cent of the ration DM as shelled corn and 20 per cent as untreated corn silage. B. Eight protein sources. (1) (2) (3) (4) (5) (6) (7) (8) Soybean meal Urea Urea + corn steep water (U-CSW - 50 per cent of N from urea and 50 per cent from natural protein) Ammonium formate Ammonium acetate Ammonium propionate Ammonium lactate Ammonium butyrate Harvesting of Feed: High moisture shelled corn axui corn silage were harvested from a stand of hybrid corn averaging approximately 35 metric tons of 35 per cent DM SiJlage or 5 metric tons of 85 per cent DM shelled corn per 1'1e<:tare. The corn silage received no additives and was 57 harvested during a three week period beginning September 2, 1971. It was stored in a 9.1 m. x 18.3 m. silo and averaged 33.4 per cent DM during harvest. The high mois- ture shelled corn was harvested in late September and early October, averaged 70.3 per cent DM at harvest and was placed in a sealed storage unit until feeding. Production of Nitrogen Supplements: The compo- sitions of the supplements used in this experiment are shown in Tables 6 and 7. All nitrogen supplements except soybean meal were added to rations as liquids. The ammonium salts were produced by neutralizing solutions of the respective organic acids with anhydrous ammonia which was introduced through a sparger system. Preliminary preparations of salts were first made in the laboratory to determine the desirable level of ammonia with the various ammonium salts. An 8.4 per cent level of nitrogen was decided upon because the ammonium salts remained in solution for all organic acids utilized. In addition, this provided a supplement that would meet the supplemental nitrogen requirements when fed at 454 g. per head daily. The ammonium salts were produced in bulk by using a 1893 l. tank equipped with a sparger system for anhydrous ammonia, an agitator, a pH probe and a water jacket for cooling. Anhydrous ammonia was introduced into the organic acid-water mixture until the pH reached 7.0. Before 58 Table 6."Experiment II: Composition of supplements used in Experiment II.l % Protein2 Supplement % Acid % DM Soy -- 50.0 85.0 Urea -- 53.9 19.5 Urea-CSW 13.0 41.9 54.6 NH4 formate 28.7 54.6 39.3 NH4 acetate 38.1 55.6 48.9 NH4 propionate 47.0 55.6 57.8 NH4 lactate 53.9 52.5 64.1 NH4 butyrate 54.3 54.1 64.8 lCalculated by using 1 mole acid + 1 mole NH3 = 1 mole salt. 2Obtained by % nitrogen x 6.25. Table 7.--Experiment II: Ingredient composition of urea - corn steep water supplement. Major Constituents % PPM Nitrogen 6.7 Lactic acid 13.0 Fat 0.2 Ash 8.5 Carbohydrates, as dextrose 1.3 Potassium 2.2 Magnesium 0.7 Sodium 0.10 Phosphorus 1.65 Sulfur 0.35 Calcium 0.03 Chlorine 0.35 Iron 150.0 Copper 50.0 Selenium 0.4 Manganese 25.0 Molybdenum. 1.0 lDerived by taking 92.7 per cent of corn steep water analysis (wet basis). 2Parts per million or grams per kilogram. 59 removal from the tank, the solution was sampled and ana- lyzed by micro-Kjeldahl to establish the nitrogen content which was then adjusted, if necessary, by the addition of water or anhydrous ammonia. A second sample was taken to establish the final nitrogen concentration and the supple- ment was stored in 208 l. barrels lined with polyethylene. The nitrogen and organic acid levels of the ammonium salts are shown in Table 6. A liquid urea supplement was produced by dissolving crystalline urea in water. Micro-Kjeldahl analysis es- tablished that the corn steep water solution contained 3.61 per cent nitrogen. To produce a nitrogen supplement with one-half its nitrogen from natural protein (corn steep water) and one-half from urea, 119.9 kg. of urea were dissolved in 1523.6 kg. of corn steep water. Table 6 shows the DM, acid and nitrogen content of the urea-corn- steep-water (U-CSW) solution and Table 7 lists the other ingredients in the U-CSW supplement. The grams of nitrogen supplement fed daily per steer were 508, 599, 463, 463, 454, 463, 481 and 463 on a wet basis for soy, U-CSW, urea, ammonium formate, ammonium acetate, ammonium propionate, ammonium lactate and ammonium butyrate treatments respectively. Feed Analysis: Silage and shelled corn samples were taken weekly throughout the feeding trial. Composite samples of fresh silage or shelled corn were analyzed for 60 nitrogen and organic acid fractions and then expressed on a dry matter basis from analyses of paired samples dried at 55° C. for 48 hours. Aqueous extracts of feeds were prepared by homogenizing a 25 g. aliquot of the feed and 100 ml. of distilled water with a Sorvall Omni-Mixer for two minutes. A portion of the unstrained homogenate was used to determine total nitrogen by micro-Kjeldahl procedures. The analyses for pH, water soluble NPN, ammonia, volatile fatty acid and lactic acid of the feeds were conducted using the procedures described in Experiment I. Chemical analysis of corn silage and high moisture corn fed during the trial are averaged in Table 8. Feeding Trial: A total of 160 yearling Hereford steers purchased in Lusk, wyoming from two consignors were utilized in this experiment. They arrived at the MSU Beef Cattle Research Center on October 8 and were fed free choice corn silage plus soybean meal for 28 days before being placed on the experiment at an average weight of 358.2 kg. Pre-experimental performance is shown in Appendix I. All cattle were full fed twice daily a ration of either 60 per cent corn silage and 40 per cent concen- trates or 20 per cent corn silage and 80 per cent concen- trates on a dry matter basis, as outlined in Table 9. 'Steers receiving the urea supplement were fed an additional Table 8.--Experiment II: feeds fed.1 61 Average chemical analysis of Corn High Moisture Observation Silage Sh. Corn Percent Dry matter 35.60 70.89 Crude Protein Fractions: Total crude protein 7.44 10.74 Organic protein 6.25 8.44 NPN protein 1.19 2.30 Ammonium salts .06 .21 Urea .06 .00 Unidentified 1.07 2.09 Organic Acid Fractions: Total organic acid 7.57 1.86 Lactic acid 5.43 1.79 Acetic acid 2.14 .07 Butyric acid .00 .00 pH 4.27 4.83 1Each value is the mean of analysis conducted each week during the feeding trial. .coauflmomaoo How u ocm m moanma mom 62 m .cOHuHmomEoo you w mance comm .GOflUflHDEHOm HON OH QHQMB mmmH m.m H.m m.an ~.H~ manususn smz an m.m m.m m.a~ m.H~ mumuoma amz ma m.m v.~ v.~h s.a~ mumcoflmoum «m2 ma o.v ¢.~ o.~h m.H~ mumumom «m2 Ha h.m m.H ~.mn m.H~ mumsuom smz m m.m o.H m.mn m.H~ «mus a m.m o.m m.Hp m.a~ mzmoua m m.m m.v m.on H.H~ som m mmmaflm CHOU ucmo mom om can mumnucmocoo ucmo umm om m.m m.m s.mm H.mm mumnmusn emz ma e.m H.m H.mm o.mm mumuoma 6.32 «H m.m m.m m.mm «.mm mumcoflmoum emz NH v.m H.~ m.mm s.mm mumumom emz oa m.m m.H h.mm m.mm mumsuom vmz h m.m o.H 4.6m m.mm mm“: m m.m m.~ m.mm «.mm mzmono m m.m v.v n.4m «.5m mom H momafim cuou ucmo mom cm can mumuucmocoo ucmo mom ow ucmsmammsm ucmfimflmmmm _ cuou mmmHflm ucmfimammdm .oz Hmumcfiz Han cflmuoum cmaamnm cuoo cwmonusz you H N .mfimmn SQ m so cofluwmomfioo coflumm "HH ucmEfiHmmeIl.m magma 63 56.8 g. (fresh basis) daily of high moisture shelled corn to make the energy in the urea rations equivalent to that in the soy ration. The organic acid in all other supple- ments was assumed to furnish equal energy to that contained in the soy supplement. The steers were fed 18.6 mg. of diethylstilbestrol (DES) daily and vitamin A and D in the mineral supplement (Table 10) which was withhely during the final seven days of the experiment. The corn silage and shelled corn averaged 35.6 per cent and 70.9 per cent DM during the feeding trial, respectively. The shelled corn was rolled and all ration ingredients were combined and mixed in a horizontal mixer just prior to each feeding. The experiment was initiated on November 2, 1971. Steers were randomly alloted by weight to the 16 treatment groups shown in Table 7. Final weights were taken on March 9, 1972 after 124 days on feed. All cattle were then trucked 161 km., allowed to stand overnight and slaughtered the next day. After 48 hr. in the cooler, carcasses were ribbed, graded by a federal grader and carcass measurements taken as described in Experiment 1. Experiment III--Acetic and Lactic Acid Additions to a High Concen- trate Ration Design: A 3 x 2 factorial design involving 36 Herford-Angus crossbred yearling steers was used to study the following treatments: 64 Table lO.--Experiment II: Formulation of mineral supple- ment. Ingredient % Dicalcium phosphate 6.0 (20% c - 18.5% P) Calcium carbonate 8.5 (38% Ca) Sodium sulfate 3.9 (22.5% S) ‘ Trace mineral salt 11.0 (High Zn) Ground shelled corn 69.2 Stilbosol (Zg/lb) 1.0 Vitamin A (30,000 IU/g) 0.2 Vitamin D ( 9,000 IU/g) 0.2 TOTAL 100.0 A. Two sources of supplemental nitrogen (l) Soybean meal (2) Urea B. Both sources of supplemental nitrogen were com- pared with different acid additions to the ration. (1) Control - no acid added (2) Acetic acid addition (3) Lactic acid addition The soybean meal and urea supplemented controls (no acid addition) were the same lots that were used as controls in EXperiment I. 65 Harvesting of Feed: The steers on this experiment were fed the same untreated corn silage and high moisture shelled corn described in Experiment I. Feed Analysis: Feeds were sampled and analyzed as in Experiment I. The average chemical analysis of corn silage and high moisture corn fed during the trial are shown in Table 3. Feeding Trial: Steers utilized in this experiment originated in Texas and were purchased as fall calves on July 22, 1970, at an average weight of 253.8 kg. They '4 were placed in outside lots and fed a full of corn silage until they were placed on this experiment and Experiment I, November 3, 1970, at an average weight of 294.9 kg. The various treatments were compared on a ration composed of 25 per cent untreated corn silage and 70 per cent concentrates on DM basis (Table 11). The soy- bean meal and urea were combined with minerals and fed as dry supplements (Table 5). The acids were fed at the same molar levels as the ammonium salts in Experiment I. The steers were implanted initially with 24 mg. of diethylstilbestrol (DES) and injected with 2,000,000 I.U. of vitamin A. Four months later, all steers were reimplanted with 36 mg. of DES and re-injected with 2,000,000 units of vitamin A. The steers were weighed as described in Experiment I. The feeding trial was terminated after 170 days on feed. 66 .H unmefiummxm cw mumuomH vmz ca owom UHDUMH mo Hm>mH HmHoz .H ucmeflummxm CH mumwmom wmz ca Uflom oaumom mo Hm>mH HMHOE .GOADMHSEHOM mom m mance mom m N H oo.ooa oo.ooa oo.ooa oo.ooa oo.ooa oo.ooa AfiBOB va.mw mm.mm mm.mm ma.v~ mv.v~ oo.m~ mv.m om.m «m.m va.mm mm.mw mm.mm ma.v~ mv.v~ oo.mN mpflom Ofluomq Npflom oaumod H.mmsm mono H.mm=m mom cuoo omaamnm mmmaflm CHOU UHDOMA oaumod msoz owuomq Ofluwom mcoz wm©U< Ufiofi own: How: cmwnwom ucmfimammdm cummuuwz .mflmmn Hmuume mud m so cowuwmomEoo COHumu ucmo Hum "HHH #COEflHOQMMII.HH OHQMB 67 The cattle were utilized in a metabolic study (to be discussed later) for nine days and then trucked 161 km., allowed to stand overnight, and slaughtered during the next morning. After 48 hr. in the cooler, carcasses were ribbed, graded by a federal grader and carcass measurements taken as described in Experiment I. Experiment IV--Acetic and Lactic Agid Additions to All Silage Rations Design: Two simultaenous feeding trials were conducted to study the effect of adding graded levels of acetic or lactic acid to corn silage rations supplemented with soybean meal. Acetic acid additions were 0, 1.5, 3.0, 4.5 and 6.0 per cent of silage DM; and lactic acid additions were 0, 2.5, 5.0, 7.5 and 10 per cent of silage DM. A single group acted as controls (no acid addition) for treatments with both acids. Harvesting of Feed: Silage was the same that is described in Experiment II. Feed Analysis: Feed samples were analyzed by the procedures discussed in Experiment II. Feeding Trial: Ninety Hereford steer calves purchased at feeder calf sales in Virginia were utilized in this experiment. They arrived at the MSU Beef Cattle Research Center October 13-15, 1971, and were fed hay, corn silage to appetite plus 454 g. of 50 per cent crude protein 68 soybean meal daily. Hay feeding was discontinued on October 27, 1971 and the steers were placed on experiment November 10, 1971, at an average weight of 249 kg. Pre- experimental performance is shown in Appendix I. All cattle were full fed twice daily the corn silage ration, their allotted quantity of acid and approxi- mately 908 g. of a protein-mineral mixture (Table 12). This supplement was formulated to provide the supple- mental mineral and vitamin requirements for the steers as well as diethylstilbestrol (DES). The ration ingredients were combined and mixed in a horizontal mixer just prior to each feeding. The acids were considered 100 per cent DM and enough water was added with each increasing level of acid addition to maintain a ration DM equal to the control group. Ration composition on a DM basis is shown in Table 13. The experiment was terminated after 124 days on feed and the cattle were then used in other experiments. Experiment V--Study of Rumen and Blood Parameters Resulting from Addition of Ammonium Salts or Organic Acids to Cattle Rations Design: Rumen and blood parameters were examined for steers on Experiment I and Experiment III. Management: The cattle on Experiments I and III were held for nine days following the termination of the feeding trial for this study. The cattle were trained to 69 Table 12.--Experiment IV: Formulation of 714 supplement. Ingredient % Ground Shelled Corn 15.17 Soybean Meal 70.15 (49% CPE) Ground Limestone 1.35 (38% C) Dicalcium Phosphate 6.15 (20% c - 18.5% P) Trace Mineral Salt 6.65 (High Zn) Stilbosol (2 g./1b.) 0.37 Vitamin A (30,000 I.U./g.) 0.08 Vitamin D ( 9,000 I.U./g.) 0.08 TOTAL 100.00 consume their ration within two hours on the first seven days by removing the feed two hours after it was offered. On the eighth day, rumen samples were obtained by stomach pumping and jugular blood was sampled before feeding (To) and 2.5 hours, 5 hours and 10 hours post-feeding. The cattle were shipped to slaughter on the ninth day. Sample Collection: Rumen contents were sampled by using a one-quarter horsepower vacuum pump, a five- eighths inch stomach tube and a vacuum flask. A positive pressure was placed on the stomach tube while it was being m oo.ooa.oo.ooa 00.00H oo.ooa oo.ooH oo.ooa oo.ooa 00.00H 00.00H A4809 av.0H. bN.m mo.m mm.N IIIIIIIIIIIIIIIIIIIIIIIII Uflod Ofluumq .................... mm.m hm.w ma.m mm.a In--- Baum oflumoa .mmnm mm.mH om.mH mm.va om.ma om.va Hm.ma mm.ma HN.MH H5.mH Hmuwsflfilmom mm.ms ms.ms mm.om ~m.mm as.ms ma.om mm.~m s~.mm mm.mm mmmHHm cuoo wo.9H wm.h wDWM11‘ mm.m wo.m wm.w wo.m wm.H HOHHGOU Uflud owuomq vmwwd Uflod owuood wmwwd .mflmmn Hmupma mum m so coauwmomfioo seeps“ ucmo mom ">H ucmfiwummeIl.mH manna 71 passed down the esophagus to avoid collection of saliva. A strainer was placed on the hose and the rumen fluid was drawn into the vacuum flask. Blood samples were collected from the jugular vein with a 10 cc. syringe and a 3.81 cm., 16 gauge needle. Collection time varied from three to five minutes per steer. After collection, the blood samples were placed in test tubes with heparin to prevent blood clotting. Rumen samples were strained through two layers of cheesecloth and 1 ml. of mecuric chloride (saturated) was mixed with 19 ml. of the strained rumen fluid. Five m1. of the above mixture were then added to 1 ml. of metaphosphoric acid and centrifuged at 10,000 r.p.m. for 10 minutes. The supernatant was retained for volatile fatty acid analysis. Laboratory Analysis: Rumen volatile fatty acid analyses were determined by injecting samples of the supernatant into a Packard gas chromatograph. Blood was centrifuged at 6,000 r.p.m. for 10 minutes, and the plasma recovered with a Pasteur pipette. Urea content of the plasma and rumen ammonia were deter- mined by the micro-diffusion method of Conway (1950). 72 Experiment VI--Nitrogen Balance Study gith Ammonium Acetate, Ammonium Lactate, Urea and Soybean Meal Design: A 4 x 4 latin square design was utilized in this study. Four l8-month old cannulated Hereford steers that had an average initial weight of 379.1 kg. were fed rations containing the nitrogen supplements shown in Table 14. Treatments were randomized by time and animal as shown in Table 15. Out of each 28 day period, 21 days were allowed for steers to adjust to new rations before being placed in the collection stalls. After an adjustment period of 14 hours (overnight) in the stalls, feed intake, fecal output and urine production were measured and sampled for chemical analysis over a period of six days. During the day following collection, jugular blood and rumen fluid samples were taken immediately before feeding and at two hour intervals thereafter up to 10 hours, post-feeding. The experiment was conducted con- currently with Experiment I. The study was initiated on January 31, 1971, and completed on May 5, 1971. Feeding Regime: Steers in the collection stalls were fed twice daily at 8 a.m. and 5 p.m. The various nitrogen supplements were compared on a ration composed of 75 per cent concentrates (high moisture shelled corn, nitrogen supplement and mineral supplement) and 25 per cent untreated corn silage on a DM basis. The rations were mixed with the respective treatments in Experiment I and 73 Table l4.-—Experiment VI: Metabolic study treatments utilized. Ration Nitrogen Supplement A Soybean Meal B Urea 'C Ammonium Acetate D Ammonium Lactate Table 15.--Experiment VI: Design of experiment. Period 870 897 888 981 --------- Ration - - - - - - - - l B A D C 2 A C B . D 3 C D A B 4 D E C A 74 collected from the mixer just prior to feeding. The ration composition is shown in Table 4 and the average chemical analysis of the feeds fed is shown in Table 3. The production and composition of the ammonium salts used are discussed in Experiment I. The compositions of the mineral supplement fed with the ammonium salts, the soybean meal supplement and the urea supplement are shown in Table 5. Representative samples of all rations were taken just prior to feeding for chemical analysis and dry matter determination. Feed not consumed was weighed, sampled and discarded prior to the 8 a.m. feeding. Sample Collection: Total feces were allowed to pass through a steel grid in the floor immediately behind each steer and were collected in large plastic containers in a pit below the collection stalls. Feces were removed every morning and total output was weighed. A 5 per cent aliquot was retained each day for nitrogen determination, a 100 9. sample was analyzed daily for dry matter content and the remaining feces were discarded. At the end of the six-day collection period, all samples from each steer were thoroughly mixed, composited and sampled for immediate total nitrogen determination. Total urine was collected in a plastic carboy (in the pit below the collection stalls) which contained 200 m1. of 18 N sulfuric acid. The carboy was emptied daily 75 and urine volume was measured, then diluted to 12 liters with water and a 10 per cent aliquot was stored in a cooler. The remaining diluted urine was discarded. After the six-day collection period, all urine samples from each steer were mixed, composited and sampled for immediate total nitrogen determination. Rumen contents were sampled through permanent rumen cannulas. Rumen samples were processed and analyzed as discussed in Experiment v, Jugular vein samples (10 ml.) were taken with a 16 gauge needle. Plasma was kept for urea analysis as dis- cussed in Experiment V. Laboratory Analysis: Dry matter of feed and feces were determined daily by drying at 105° C. for 24 hours. Total nitrogen determination of feed, feces and urine were analyzed by macro-Kjeldahl procedures on fresh samples. Experiment VII--Nitrogen Balance Study Comparing various Ammonium Sglts of Organic Acids with Cpnventional Nitrogen Supplements Design: Twenty-four Hereford steers were utilized in a nitrogen balance study to compare soybean meal, 1/2 liquid urea - 1/2 corn steep water (U-CSW), liquid urea, ammonium formate, ammonium acetate, ammonium propionate, ammonium lactate and ammonium butyrate as shown in Table 16. Each supplement was fed to three steers and steers 76 Table 16.--Experiment VII: Design of metabolic experiment comparing various nitrogen supplements. Nitrogen Period Supplement 1 2 3 Soybean meal 473 445 391 U-CSW 286 302 253 Urea 301 221 386 Ammonium formate 433 272 470 Ammonium acetate 285 202 247 Ammonium propionate 317 368 394 Ammonium lactate 250 332 465 Ammonium butyrate 432 395 335 were used only once. The steers were short yearlings that had been purchased the previous fall in Virginia feeder calf sales. They were full fed an all silage ration which was treated with urea-mineral at time of ensiling from October, 1971, until the experiment was initiated. A11 24 steers were selected for uniformity in weight prior to initiation of the experiment. They were then divided according to weight into three groups to make the cattle within each group as uniform as possible. The average initial weight for each group was 404.6, 395.8 and 387.9 kg. for periods 1, 2 and 3, respectively. The respective periods were initiated on April 12, April 20 and April 28, 1972. Each period consisted 77 of 14 days in which the steers were acclimated to their ration and seven days in the collection stalls. The steers were moved to the collection stalls the night before collections were to begin. Feed feces and urine were measured and sampled for chemical analysis over a period of six days. During the day following collection, rumen fluid and jugular blood samples were taken immediately before feeding and at two hour intervals thereafter up to 10 hours, post-feeding. Feeding Regime: Steers in the collection stalls were fed twice daily at 7:30 a.m. and 5:30 p.m. They were watered three times daily from plastic buckets at 7 a.m., 12 noon and 5 p.m. The nitrogen supplements were compared on a 60 per cent untreated corn silage and 40 per cent concentrates (dry, shelled corn, nitrogen supple- ment and mineral supplement) on a DM basis. The corn silage was removed from the silo and mixed with rolled corn in a horizontal batch mixer once each day. The mineral and nitrogen supplement was thoroughly mixed with the silage-corn mixture by hand each afternoon before feeding. One-half of the daily ration for each steer was fed the same evening and the remainder was stored in a plastic bag until being fed the following morning. The steers were fed ad libitum during the first 10 days of the adjust- ment period and 90 per cent of their ad libitum con- sumption for the remainder of that period. I a .1;:-:- 7.3 Mineral supplement (Table 10) was fed at 454 g. per head daily; and the nitrogen supplement at 508, 599, 463, 454, 463, 481 and 463 g. for steers on soy, U-CSW, urea, ammonium formate, ammonium acetate, ammonium propionate, ammonium lactate and ammonium butyrate, respectively. The production of the nitrogen supplements is discussed in Experiment II and their composition is shown in Tables 6 and 7. Representative samples of all rations were taken just prior to feeding for laboratory analysis and dry matter determination. Feed not consumed was weighed, sampled and discarded prior to the 5:30 p.m. feeding. Sample Collection: Collection of feces and urine was similar to that described for Experiment VI. A 10 per cent aliquot of the feces was retained each day for nitrogen determination and a 200 gram sample was analyzed daily for a dry matter determination. Samples of rumen fluid were taken by stomach pumping and jugular blood samples were taken with a 10 ml. syringe. The procedures for obtaining, processing and analyzing these samples are discussed in Experiment V. Laboratory Analysis: Dry matter of feed and feces were determined daily by drying in an over at 55° C. for 48 hours. Extracts of the feed and feces were prepared by homogenizing 25 g. of material and 100 ml. of distilled water with a Sorvall Omni-Mixer for two minutes. A 79 portion of the unstrained homogenate was used to determine total nitrogen by micro—Kjeldahl procedure. MicroéKjeldahl procedures were also used to determine the total nitrogen of the diluted urine samples. Statistical Analysis All data reported in this dissertation were ana- lyzed on a CDC 3600 computer at Michigan State University Computer Laboratory. Analysis of variance procedures were used in all experiments, and Duncan's New Multiple Range Test was used to test for significant differences between means. RESULTS AND DISCUSSION Experiment I--Feeding Trial Comparing Ammonium Acetate Ammonium Lactate, Urea and Soybean Meal Cattle Performance: Complete results of this experiment are shown in Table 17. Average daily gain of the cattle receiving urea was depressed 5.9 per cent below the group receiving soybean meal (791 9. vs. 841 g.). Dry matter consumption and feed efficiency were slightly depressed for the urea supplemented group when compared to the soy-supplemented group. Although not significant (P < .05) these differences are in agreement with previous work (Newland gt_al., 1961 and Helmer and Bartley, 1971). Several workers have noted a lower level of performance on urea supplemented rations and have generally attributed this to a decreased palatability of such rations (Woodward and Sheppard, 1944; Wise et_al,, 1944 and Owens ep_gl., 1968). The cattle receiving ammonium acetate as a source of supplemental protein gained 5.4 per cent faster than cattle receiving soybean mean (886 9. vs. 841 g., daily) and 12.0 per cent faster than cattle receiving urea (886 g. vs. 791 g., daily). The difference in average daily 80 81 samuoum Hmucmsmdmmsm mo mousom om.mea oo.a~a oo.om oo.ooa amusmsmammsm mo msam> m>wumamm Hm.mv Hm.mv Hm.mv Hm.m¢ scams .mx ooa Hmm umoo mmmonmw Ucm pmmm mm.m mm.m ww.m mm.m .mx .cflmm .mx “mm cmmm mucmaoammm 666m .Hmhwlu .mmwwlu .mmwwul wwhwll A4909 mm.o mm.o wm.o Hm.o ucmsmammsm cmmouuflz av.m ma.m m>.¢ mm.v suoo cmaamnm .uw hm.a mh.H mm.H mm.a momHHm supm ”sowmm .mx .ommm manna mmm. onmmw. Has. Ham. .mx .cmmm manna .>« s.sme m.mee m.m~e a.mmv .mx ..u3 Hagan .>¢ m.mm~ a.mmm m.mm~ a.mmm .mx ..u3 Hugues“ .>« m m m m mummum mcHHummh mo .02 .mH ha ow ad .02 uoq mumuomq mumumod mmHD Ham: umma awn 05H vmz fizz smmnhmm .iasma .mm Hflum< op cums .m Hansm>ozv mauumo uoHUmmm MOM samponm mpsnm mo monsom m mm muamm EUHGOEfid "H ucmfiwummeIl.nH manna 82 . So. v E 0 .Q .m “waucm0flmwcmflm umwmac umflnomnmmsm ucmummmac mcfl>mn mmsam> "mOGMOflmwsmflm .unmflm3 ucmEfiummxm mmo Hm>o unmflm3 mmmmnmo UHOO .muso Hflmumu UmEEwHu .mmmamson cw usmflm3 mummumo mo usmo Hmm o m .umM ma>amm ocm pummn .mmspwx CH unmams mmmoumo no name umm .mH .VH .MH .NHanHOSU “Ha .oH .muwoow v .ba .mHHmHMHmwOE “ma .VH .mauummvoz «NH .HH .oauaamfim m N .wmp\nmmum\ooa um mmmpumm .us\om.mwm cuoo smaflmnm us\sm.mw mmmHnm cuoo so wmm so swans mumoo swam H ma.mflam ma.maam mH.mHHm om.maaw .mx cos umm momma ammoumo sm.om ww.om om.om mam.ao Guamo umd mcfimmmno bh.hv mh.bv vm.mv Hm.hv mmuno .m .B .m ucmo Hmm em.m mo.v oo.v oo.w vumm .m .m .M ucmo umm Hm.mm oo.mm om.mm sm.mm mac «mum mam nflm m>.H mm.H mm.H mm.a .Em .mmmsxoflnu umm sm.ma so.¢a oo.ma om.ma mmuoom mandamus om.ma sa.ma Hm.MH sm.ma «momma ammoumo "sowmm5H0>m mummumu mumuomn mumumo< mmuD Hmmz ummemen oha vmz vmz cmmnmom camuoum Hmucmfimammsm mo monsom pmscwuc00|l.ha magma 83 gain was significant (P < .05) between ammonium acetate and urea but not significant between soybean meal and ammonium acetate. Varner and Woods (1969) also noted a significant (P < .05) increase in average daily gain when ammonium acetate supplemented cattle were compared to cattle fed urea. Other work with lambs fed ammonium acetate (Repp e£_213, 1955a) and cattle fed a high-acetate ammonium salt mixture (Varner §E_gl., 1968) have resulted in equal growth when compared to animals fed urea. Varner §E_§1. (1968) also reported that the high-acetate ammonium salt mixture supported growth almost equal to soybean meal for finishing cattle. The ammonium acetate fed cattle consumed 3.1 per cent more DM than the soybean meal supplemented steers and 6.0 per cent more than those fed urea. Feed efficiency was 2.3 per cent and 6.1 per cent greater, respectively for steers fed ammonium acetate than for those fed soybean meal or urea. Ammonium lactate supplemented steers had signifi- cantly (P < .05) higher average daily gains than either the soybean meal or urea fed groups. The group receiving ammonium lactate as a source of supplemental nitrogen gained 13.5 per cent faster than the group supplemented with soybean meal (955 9. vs. 841 g., daily) and 20 per cent faster than the group receiving urea (955 9. vs. 791 g.). The ammonium lactate fed steers consumed 9.1 per cent 84 more DM than soybean meal fed animals and 12.2 per cent than the urea fed group. Similarly, feed efficiency was. increased 3.5 per cent and 7.2 per cent with supplementation of ammonium lactate when compared to rations supplemented with soybean meal and urea, respectively. Performance of the ammonium lactate and ammonium acetate groups was not significantly different (P < .05). However, average daily gain, daily DM consumption and feed efficiency for the ammonium lactate group were 7.8 per cent, 5.8 per cent and 1.2 per cent higher, respectively. No feeding trials or growth studies have been reported in the literature that involved ammonium lactate. However, the superiority of ammonium lactate over other NPN sources agrees with in vitro studies by' Belasco (1954). Relative Value of Supplements: The relative value of the four supplements is shown in Table 17 and was de- rived by equating for all treatments the total feed and yardage cost per kg. of gain and allowing supplemental nitro- gen cost to vary. By assigning a relative value of 100 to soybean meal, the value of the other supplements is expressed relative to soy. This figure is an economic evaluation of the supplements based on average daily gain and feed efficiency. 85 Under the conditions of this experiment, urea had only 80 per cent of the value of soybean meal. Ammonium acetate and ammonium lactate were worth 21.0 per cent and 48.2 per cent more than soybean meal. Carcass Evaluation: Differences in carcass desira- bility were small, and for the most part, non-significant. All groups of cattle graded average Choice, possessed a modest to moderate level of marbling and yielded approxi- mately 48 per cent of their carcass weight in boneless, trimmed, retail cuts. The urea treatment group was trimmer and had highest yield of retail cuts. The soybean meal fed steers had the highest (P < .05) dressing percentage. Experiment II--Feeding Trial Comparing various Ammonium Salts of Organic Acids with Conventional Nitrogen Supplements Cattle Performance: Table 18 shows the mean per- formance of the two concentrate levels for each nitrogen supplement. The differences in performance were not sig- nificant (P'g .05). However, the average daily gain of the urea fed cattle was depressed by 5.3 per cent below the performance of the soybean meal group. This is consistent with the 5.9 per cent reduction in the average daily gain of the urea compared to the soybean meal group in Experiment I. The urea fed steers gained slower than all other groups on the low level of concentrate and slower than all but the ammonium acetate fed cattle on the high concentrate ration. 86 . . . . . . . . mucmEmHmmsm mm mm ON ON ON HHH mm mm OH ON Om mm mm Om OO OOH mo maHm> m>HmmHmm . . . . . . . . chO .OH OOH “mm OO Om» OO ONO OO ONO OO ONO OO ONO OO OH» NO ONO OO ammummo mmmmums 6cm Ommm m ON.N ON.O Om.N OO.O NO.N OO.O OO.N HO.O .chm .Ox umm mwmm ”NucmHUHmmm pmmm OO.HH HO.HH OO.OH ON.HH OO.HH HO.OH mm.HH OH.HH Hmmoa NO.O M0.0 N0.0 N0.0 N0.0 N0.0 NO.O NO.O mcmsmHmmsm HmsmcHz mm.O mm.O NN.O mN.O HN.O OH.O mm.O Om.O mcmsmHmmsm cmmoumHz OO.O NO.m Om.m OO.m O0.0 OO.m N0.0 OO.m cuoo HHmnm .uo OO.O NO.O ON.O mO.O ON.O NO.O Om.O Om.O mOmHHm cuoo "so NmO .wx .Ommm HHHmo OO.H mO.H NO.H HO.H mO.H Om.H OO.H mO.H .Ox .chO sHHmO .>m a.mOm N.HOm N.HOm O.Omm N.OOm a.mOm 0.0mm H.NOm .>< O.Omm O.Omm N.Omm m.smm p.mmm O.Omm O.Omm O.Omm .u3 HmHchH .>m mummum ON ON ON ON ON ON ON ON OcHHumms mo .02 mums mpmuomq mpm: mumumod mumfinmh, Iwusm v Immoum v v mmHD ZmUID mom Omz mz Omz mz m2 mmme smo ONH sHmuonm mosuo HmusmEmHmmsm mo mousom .HNOOH .O roams om HNOH .N H35>on mHmmmo MOHUTGM .HOM GkuOHQ OUSHU m0 OUHDOm 6 mm mun—”mm EDHGOE ”HH #CGEHHGQNMII.mH THDMB 87 .Hao. v my n «v “mHusmoHMHsmHm smumao mumflsomstSm psmsmmmwp MWW>mm MWDMMM "mosmoHMHsmHm .umoo ommm psm mosmfloflmmm pmmm m>wumHmH .sHmm mHHmo mmmsm>m m>HDMHmH so wmmmmN .usmflm3 usmEHHmmxm mmo Hm>o usmwm3 mmmoumo GHOUm .muso HHmums UmEEHHu .mmmHmsos sH usmflm3 mmmoumo mo psmo Hmmm .umm UH>Hmm psm mumms .mmspwx sH usmwm3 mmmosmm mo usmo smmO .OH .NH .OHnmmmsmmos NmH .OH .mH mmmooz NNH .HH .OHuHHmsmm .OH.MH.NHnmoHosU NHH .oH .muooowm .mmp\smmum\ooa um mmmpsmm .uE\mH.mww usmEmHmmsm Hmsmsfle ..uE\oo.mOm suom pmHHmsm ..uE\Nm.mw mmmHHm sHoo So wmm so cmmmn umoo ommmH .OH OOH mN.¢HHw ho.wHHm Hm.vaaw ma.mHHm ON.mHHm mN.MHHw mm.vHHw mN.¢HHw No. mm. 9 m5. OO. mON. ON. mOO a NO Om. NO mON N NO. ON NO. H mm. OH mNmN. HH anO NO ON. NO OHmNO. O ON. ON OO. H mOO. HH a mN. HH nmmO Om OO.Om OO anH.OO N OO.m ON ON.NN H mHHOOH HH mO.HH HH anN HH ON.Nm NO.Nm NN. nNO NO mON OO nmN mO.m ON.N mN. OH.ON mN.mN Om. ON.H Nm.H OO. mmN.HH nmm0.0 OHOO. mOO HH OHON OH anm Om OO.Om NO anm.OO N ON.N ON OO.ON H mON.H OH m0.0 HH nOOO HH Hmm mowsm mmmoumu usmo Hmm mswmmmsa mmuso .m usmo Hmm umm .m .m .M as o smm Em .mmnm mum Dam .mo .mmmsx0flgu umm mmsOOO OOHHOst NmOOsO mmOOHOo ”COMHMSHMKVW mmMUHMU w .m .B 88 As in Experiment I, the daily DM consumption and feed efficiency of the urea supplemented cattle was slightly lower (2.3 per cent and 3.2 per cent, respectively) than for the soybean meal fed steers. Gain of the groups fed the various ammonium salts was practically identical to that of the soybean meal and U-CSW groups when averaged over both concentrate levels (Table 18). However, performance on all the NPN supple- mented diets except ammonium acetate increased at the higher level of concentrate (Table 19). Cattle fed ammonium acetate at the high concentrate level gained 3.8 per cent less than those on low concentrate. The increase in gain for high versus the low concentrate rations was 2.9 per cent, 5.4 per cent, 4.2 per cent, 10.0 per cent, 2.6 per cent and 4.4 per cent for the U-CSW, urea, ammonium formate ammonium propionate, ammonium lactate and ammonium butyrate fed groups, respectively. On the high concentrate ration, all of the NPN fed steers except urea and ammonium acetate outperformed the soybean meal fed steers. In contrast, the soybean meal fed steers outgained all groups on the low level of concentrate. In addition, all of the NPN groups were more efficient than the soybean meal fed steers on the 80 per cent level of concentrate but less efficient than the soybean meal group on the low level of concentrate (Table 19). 89 ON.N HH.O NN.N ON.O mm.N ON.O ON.N Nm.N .OH .chO .OH Ommm wNmsmHowmmm ommm Om.HH Nm.HH OO.OH OO.OH mO.HH mm.HH NO.HH mN.HH Hmmoa N0.0 N0.0 NO.O NO.O N0.0 NO.O N0.0 NO.O mcmsmHmssm HmumcHs ON.O HN.O OH.O OH.O N0.0 N0.0 OO.O OO.O ucmsmHmmsm cmmoumHz OO.O HH.O OO.N OO.O OO.N NH.O Nm.N OO.m ammo HHmnm Osmoso NO.N ON.O ON.N OO.O NN.N ON.O ON.N OO.O mOmHHm :noo "so wmm xmx .ommm HHHmo OO.H NO.H HO.H NO.H OO.H NO.H NO.H NO.H .OH .chO sHHmO .>m O.OOm N.HOm 0.0mm O.ONm a.mOm a.mmm 0.0mm 0.00m .OH ..p3 Hmch .>m N.mmm m.mmm N.Omm N.Omm m.Nmm m.Nmm O.mmm O.mmm .mx..u3 HmHuHcH .>< OH OH OH OH OH OH OH OH mummum msHHummN mo .02 O N O m m m N H uoH wow wow wow wow wow wow wow wow soflumsusmosoo usmosmm mumssom vmz mmsD ZmUID mom mOHSOm smoouqu mHm>mH mumsusmosoo osm mmousom samposm mossm msoflum> mo ummmmm .HNNOH .m scum: om HNOH .N H35>on "HH usmfiflsmmeIl.mH magma 90 .usoflm3 psmsfiummxm mmo Hm>o usmfim3 mmmoumo CHOU .umm 0H>Hmm psm usmms .mmsofix sH usmwm3 mmmoumo mo usmo “mm m .muso HHmumH omEEHHu .mmmamson sfl usmfim3 mmmonmo mo usmosmm s m .OH .NH .OH mumummoz 1OH .OH .OH u ummmoz uNH .HH .OH u HHmsmN .OH .NH .NH u moHoso uHH .OH .O u moooH O0.0HHO mO.mHHm ON.NHHO ON.OHHO mm.mHHm ON.NHHO Om.mHHm OO.NHHO .mx OOH smm mOHum mmmmumo ON.OO NH.Nm ON.Om ON.Nm mm.mm mO.Nm mN.mm HN.Om mucmo smm mchmmso N0.00 Nm.OO mN.OO NN.OO NH.OO ON.NO OO.NO O0.00 Ommso .m .9 .m ammo umm OH.O mO.N om.N ON.N ON.N om.N ON.N mm.N mumm .m .m .M memo nmm OO.NN OO.NN mN.mN ON.ON OO.NN OO.HN O0.0N N0.0N N.so .mmum mam nHm mO.N Nm.H NO.H NO.H OO.H OO.H mm.H NO.H .so .mmm2HOHnu umm ON.HH OO.NH O0.0 ON.O ON.HH O0.0H OH.HH O0.0 Nmuoom mcHHnumz OO.HH OO.NH OO.OH O0.0H ON.HH ON.HH OO.HH OO.HH Hmmmum mmmosmo ”GOHHMSHM>W mmMOHMU 91 mm.N ON.O OO.N NO.O NN.N Om.N Nm.N mm.m .OH .chHO .OH smm mmmm "musmfioflwmm ommm OO.HH OO.HH ON.OH ON.HH OO.HH OO.OH NO.OH ON.NH_ Hmuos NO.O NO.O NO.O NO.O NO.O NO.O NO.O NO.O ucmsmHmmsm HmumcHz ON.O mm.O mm.O ON.O NN.O NN.O mN.O ON.O pcmsmHmmsm :mmouqu ON.N HN.O mm.N OO.O ON.N NN.N Om.N NO.O anon HHmrm museum ON.N NO.O ON.N mm.m ON.N NN.O ON.N mN.N mmmHHm csoo "so wmm «mg .Ommm NHHmo Hm.H OO.H OO.H HO.H Om.H mm.H Om.H OO.H .Ox .nHmO NHHmO .>m O.mmm m.mOm m.mOm m.Nmm m.OOm O.mmm N.mmm m.OOm .mx ..u3 HmcHH .>m O.Omm O.Omm O.Omm m.Nmm m.Nmm N.Omm N.mmm m.mmm .mx ..u3 HmHuHcH .>m OH OH OH OH OH OH OH OH mummmm OOHHsmmN mo .02 NH mH mH OH OH NH HH OH moH mom OOO mom OOO OOO OOO OOO OOO mmumsucmoaom mamosmm mumswusm vmz mumuomq vmz mumsflmosm vmz mumumms HOmz monsom smmosuflz Ill omssflus00|l.ma magma 92 .usmfim3 usmsfiummxm mmo Hm>o unmflm3 mmmoumo GHOOm .muso Hflmumu omEEfluu .mmmHmson sfl usmflm3 mmmosmo mo usmo “mm...V .umm 0H>Hmm osm usmms .>mson sH usmflm3 mmmoumo mo usmo Hmmm .OH .NH .OH u mumumwoz 13 .OH .NH u ummmoz uNH .HH .OH n HHmsmN .vH .MH .NH u mowono “dd .0H .m n GOOOH ON.mHHO OO.NHHO NO.NHHO NH.mHHm ON.OHHO OO.OHHO Om.mHHO ON.OHHO .OH OOH umm moHsm mmmoumo mv.mm N®.mm mm.mm Ha.mm ma.mm mm.bm hm.mm v0.5m musmm Hmm mswmmmuo mH.h¢ Nh.mv mm.hv mo.wv ¢H.mv HH.mv m®.hv Oh.mv vmuao .m .B .m usmo Hmm OH.m mm.m oa.m mm.N oo.m oo.m mm.m mo.N mumm .m .m .M usmo Hmm vm.¢h mm.wb No.0h 00.05 vo.mb mm.¢h mm.hh Nv.bb NED .mmum mam nflm om.H oo.a mm.H om.H om.H ov.H mm.H mo.H .50 .mmmsxownu “mm OH.NH om.m O0.0H om.NH om.HH ow.OH om.NH om.m NmHOOm msflanumz ON.NH 00.0H om.HH om.NH OO.NH om.HH OH.NH om.HH Hmwmum mmmoumo u GOflHflSHflNrW wMMUHMU 93 These results do not support the theory that the increased energy availability of the ammonium salts is responsible for greater nitrogen utilization. A possible explanation for the increased level of performance on the high concentrate diet with the NPN supplements is an in- creased urea fermentation potential (UFP) of the ration as discussed by Burroughs §E_gl. (1972). Relative Value of Supplements: The relative values of the supplements are shown in Table 18, and the deri- vation of this figure is discussed in Experiment I. Under the conditions of this experiment, ammonium propionate had the greatest value (112 per cent of soy) and urea had the least value (36 per cent of soy). Therefore, a feeder could pay 3.1 times the cost of urea for the ammonium prOpionate supplement or 2.8 times the cost of urea for the soybean meal supplement and not affect his net return. Using this system of evaluation, the decreasing order of value for the supplements was ammonium propionate, soybean meal, ammonium butyrate, ammonium lactate, ammonium formate, U-CSW, ammonium acetate and urea. The relative value of the NPN supplements increased on rations of 80 per cent concentrate and decrease at 40 per cent concentrate. Belasco (1954) did not test soybean meal, U-CSW, or ammonium salts of propionic and butyric acid. However, he did report the following decreasing order for nitrogen utilization, ammonium lactate, ammonium formate, urea and ammonium acetate. This is practically the same order in 94 which these supplements are listed for relative value. Belasco's rank on in vitro cellulose digestion does not follow this order and ammonium acetate fed steers out- performed those fed urea in Experiment I. Carcass Evaluation: When averaged across both levels of concentrate (Table 18), the carcass grade for the urea supplemented cattle was average good and the average carcass grade for all other groups was high good. The urea fed steers graded significantly (P < .05) lower . than the ammonium formate cattle and the difference in carcass grade approached significance when the urea cattle were compared to cattle fed ammonium salts of lactic, acetic and propionic acid. The cattle on this experiment had an average final weight of 541.8 kg. and average fat thickness of 1.72 cm. However, only 50 per cent of the cattle graded Choice. In contrast, the cattle on Experiment I were lighter (443.3 kg.) at slaughter and had approximately .the same fat thickness (1.64 cm.) but 96 per cent graded Choice or better. The low carcass grades on this experi- ment may have been of genetic origin. This hypothesis is supported by the fact that steers originated fromHonly two herds (Appendix I). Carcass grade, on the average, was significantly (P < .05) higher for the higher level of concentrate (Table 20). However, increased carcass grades on 80 per cent 95 concentrates were not observed for all of the nitrogen sources tested. Steers supplemented with urea, ammonium formate and ammonium lactate had lower carcass grades on the high concentrate ration. The interaction between nitro- gen source and concentrate level for carcass grade was significant (P < .01). This same trend existed for marbling score, which is the primary determinant of 12‘ “1‘... carcass grade. The urea fed steers not only yielded lower grad— ing carcasses but also had less fat and lower carcass price than all other groups. Carcasses from the urea fed cattle were the trimmest in all fat measurements (external fat and internal fat), as well as in all carcass measurements that are correlated with the amount of carcass fat (marbling score, cutability and dressing per cent) as shown in Table 18. Difference in fat thick- ness was not significant (P < .05) between the urea and ammonium propionate fed cattle but it was significant (P < .05 or P < .01 as shown in Table 18) between the urea cattle and each of the other treatment groups. The urea fed cattle had the lowest marbling score and this difference approached significance (P = .062). Carcasses from the urea fed cattle had signifi- cantly (P < .05) greater yields of boneless, trimmed retail cuts than carcasses from cattle fed U-CSW, ammonium formate, ammonium lactate and ammonium butyrate. 96 Carcasses from the urea fed cattle in Experiment I also had less external fat and a greater yield of boneless, trimmed retail cuts than those from other treatments, but the differences were not significant. The differences in all other carcass traits when averaged across both concentrate levels (Table 18) among the steers fed ammonium salts, soybean meal or U-CSW were small and nonsignificant. Concentrate Levels: Results of this comparison are shown in Table 20. The 40 per cent concentrates - 60 per cent corn silage and the 80 per cent concentrates - 20 per cent corn silage rations are approximately equal to 70 per cent and 90 per cent concentrate in the ration, respectively. Although not significant (P < .05), the average daily gain was 2.9 per cent higher (1.45 vs. 1.41 kg.); and the feed required per unit of gain was 7 per cent lower (7.54 vs. 8.11) for the high concentrate diet. However, the lower concentrate ration resulted in more beef production per hectare (1,348 vs. 1,039 kg.), a higher gross return per hectare ($892 vs. $702) and a lower cost per 100 kg. of gain ($36.34 vs. $41.60). Compared to cattle fed low concentrates, those on high concentrates had a significantly higher carcass grade (P < .05) and dressing per cent (P < .01). More- over, carcasses were significantly fatter (P < .01) as 97 indicated by both external fat and per cent of the car- cass weight as kidney, heart and pelvic fat. In addi- tion, feeding the high concentrate ration resulted in carcasses that had significantly lower (P < .01) yields of boneless, trimmed retail cuts. Experiment III-—Acetic and Lactic Acid Additions to High Concentrate Rations Cattle Performance: As shown in Table 21, add- ing acetic acid to an otherwise nutritionally balanced ration had no effect on average daily gain, but adding lactic acid stimulated gains 3.4 per cent (840 g. vs. 813 g.). The addition of acetic or lactic acid to the urea supplemented ration resulted in faster growth (Table 22), but only lactic acid addition was beneficial when added to soybean meal supplemented rations. The growth promoting effect of added organic acids, especially to urea supplemented rations, could be a result of increased energy for fermentation as reported by Arias gE_gl. (1951) or it could be that the acid radical decreased the absorption of ammonia into the blood as suggested by Wetterau and Holzchub (1960 and 1961) and reported by Coppock and Stone (1968). The addition of either acetic or lactic acid did not depress DM consumption as has been cited in the literature (Simpkins et al., 1965; Weston, 1966; Rook 98 Table 20.--Experiment II: 40 per cent shelled corn and 60 per cent corn silage vs. 80 per cent shelled corn and 20 per cent corn silage (November 2, 1971 to March 9, 1972). 128 Day Test 40% Sh' corn aggr: . 60% Silage 20% silage No. of yearling steers 80 80 Av. initial wt., kg. 358.2 359.1 Av. final wt., kg. 538.4 544.8 Av. daily gain, kg. 1.41 1.45 Daily Feed, kg. 85% DM: Corn silage 6.67 2.35 Ground shelled corn 4.06 7.90 Nitrogen supplement 0.30 0.30 Mineral supplement 0.42 0.42 Total 11.45 10.97 Feed Efficiency: Feed per kg. gain, kg. 8.11 7.54 Feed and yarda e cost per 100 kg. gain? $36.34 $41.60 Beef produced/hectare corn fed, kg. 13.48 10.39 Gross returns/hectare corn fed $892 $702 Carcass Evaluation: Carcass gradez3 11.33a 11.69 Marbling score 10.35A 11.00 Fat thickness, cm2 1.60 1.83 Rib eye area, cm. 4 75.29A 76.13 Per cent K. H. A. fat 5 2.72A 2.98 Per cent B. T. R. cuts 48.58A 47.82 Dressing per cent 57.84 58.86 Carcass price per 100 kg. $114.29 $114.62 1 2See Table 18 for feed cost. 3Good = 9, 10, ll; Choice = 12, 13, 14. Small = 10, 11, 12; Modest = 13, 14, 15; Moderate = 16, 17, 8. Per cent of carcass weight in kidney, heart and pelvic fgt. Per cent of carcass weight in boneless, trimmed retain cats. 7Cold carcass weight over off experiment weight. Based on corn yields of 35 mt. of 35% DM silage or 5 mt. of shell corn per hectare. 8Based on selling price of cattle. Significance: Values having different superscripts differ significantly; A = (P < .01), a = (P < .05). 99 OH.HOH oO.MN oo.om Hmusmsmammsm mo msHm> m>flumHmm Hm.mO Hm.mv Hm.ov Hsflmm .mx ooH smm umoo monoumm osm ommm ov.m Ho.m oo.m .mx .swmm .mx smm Ummm “moamHOHmmm Omms NH.N mm.N no.N Hmuoe mm.o mm.o mm.o usmEmHQQSm smmosuflz Hm.v mo.m mw.v suom UmHHmSm .Hw mo.H NN.H mo.H mmmHHm suoo _ "so NmO .mx .Ommm NHHOO ovw. mam. mam. .mx .sflmm maflmo .>¢ a.mmv m.ovv N.omv .mx ..p3 Hmsflm .>< m.omm v.mom N.mmm .mx ..u3 Hafiuflsfl .>¢ HH HH NH msmmum msHHHmmm mo .02 oauomq owpmos Hosusoo umme awn oNH Uwom manoma osm cams owumom mo uommmm .HHNOH .NN HHsmm op ONOH .m umnsm>ozo :onHOmm "HHH usmEHsmmxm14.Hm magma .Hmo. v mV n .m NmHusmoHMHsmHm HmMMHo umHHomstsm usmHmMMHo msH>ms mmsHm> "mosMOHMHsmHm usmHmB usmEHHmmxm mmo Hm>o usmHmz mmmmumo CHOUo .mpso Hngms UmEEHHu .mmmHmson sH usmHm3 mmmonmo mo usmo smm .umM 0H>Hmm osm “Hum: .mmsUHx sH usmHm3 mmmoumo mo usmo Hmm .OH .NH .OH n mmmumuoz “OH .OH .OH n ummmos mNH .HH .OH u HHmsm Md'ln .OH .OH .NH u mOHono uHH .OH .O n 6006 100 uE\Nm.mw mmMHHm ssoo So usmo smm mm so ommmn mumoo ommm N .mmo\smmum\ooH pm masons» .uE\oo.mOw ssoo omHHmsm H mm.MHHm Hm.OHHm Hm.OHHm .mx ooH Hmm moHHm mummsmu NN.Ho Om.Ho oo.Ho ousmo Hmm msHmmmso hm.mO mo.mO NH.mO mmuso .m .9 .m usmo smm soO.m Om.m oo.O Oumm .m .m .M usmo smm NN.OO NO.HN N0.00 N.so .mmsm mam nHm Nm.H mm.H mm.H .Eo .mmmsmesu umm ON.NH N0.0H mN.mH mmnoom mcHHnsmz mH.NH om.MH NO.MH momsm mmmosmu m usOHumsHm>m mmmosmo .i, Ill-l ( O0.00 O0.0N O0.00 OO.OOH ON.NN OO.OOH HmucmsmHmmnm mo msHm> m>HumHmm Hm.mO Hm.OO H0.00 HN.OO H0.00 H0.00 chO .OH OOH smm umoo mmmmums mam mmmm mm.m NH.m om.m vm.m om.m mm.m .mx .sHmm .mx Hmm Ummm nwusmHonmm ommm O0.0 HO.N O0.0 NN.N ON.N mH.N Hmuoa mm.O Om.O O0.0 Nm.O N0.0- Hm.O ucmsmHsmsm cmmouqu NN.O NH.m ON.O OH.m N0.0 OO.O cuom OmHHmcm .uo HO.H mN.H NO.H mN.H Om.H OO.H mmmHHm :soo "so Nmm .OH .Ommm NHHmo NHO. mom. OON. Nmm. NNO. OOO. .OH .chO NHHmO .>m N.mmO m.mmO m.OmO m.mOO H.NOO m.OmO .OH ..H3 Hmch .>< O.NON O.HOm m.OOm O.mON N.mOm m.mON .OH ..m3 HmHchH .>m m o o o m o msmmum msHHsmmm mo .02 OH HN ON OH NN OH .08 uoH mHom OHom mHom OHom mHuomq OHumos Houusoo UHUOMH 0Humo< Houusoo umms mmo ONH usmEmHmmsm ammo usmEmHQQSm mom .HHNOH .NN HHumm on ONOH .m smasm>ozo msoHums omusmEmHQQSm mmss osm mom on soHuHUUm UHom 0HuomH osm oHom 0Humu¢ .HHH usmEHHmmxmsl.Nm mHnt 102 \Hmmum\00H um masons» ..uE\oo.avw ssoo omHHmsm ..us\hm.aw maMHHm ssoo so wmm so ommmn mumoo ommm .muso HHmumu omEEHHu .mmmHmson sH usaHmz mmmosmo mo usmo umm .umm 0H>Hmm osm unmms .mmson sH usaHm3 mmmosmo mo usmo Hmm .mH .hH .mH u .unaHmz usmEHummxm mmo um>o usaHms mmmosmo oHOO mumummoz “OH .OH .OH u ummooz NNH .HH .OH n HHmsm .VH .MH NNH n mOHono NHH .OH .a u oOOU m m o m N .mmo H oh.MHHm Hv.om w~.av Hm.m vb.h® hH.H HO.HH mm.HH om.mHHw hm.H® Ha.mv oo.v mm.Hh ov.H 5H.mH mm.MH MH.mHHm om.om om.mv oo.v oa.o® mm.H oo.mH om.MH NN.¢HHm MH.N® wv.mo Nv.m om.oa om.H om.vH mm.NH Nm.mHHw Hm.Ho om.wo am.m @O.Nh mo.H am.oH 5N.MH m¢.mHHm mm.H® Hm.hv oo.v oa.mm Oh.H om.mH mm.MH .ax ooH umm mOHHm mmmoumo ousmo Hmm asHmmmso mmuso .m .9 .m usmo smm Oumm .m .m .M usmo smm N.Eu .mmum mam QHm .80 .mmmsonsp umm mmuoom asHHnsmz Nmomma mmmosmu u GOHHM5HM>N mmMUHMU 103 gg_gl., 1963; Montgomery gt_gl,, 1963 and Baile and Pfander, 1965). Acetic acid depressed feed efficiency 3.7 per cent (9.01 vs. 8.69) but lactic acid increased feed efficiency 2.6 per cent (8.46 vs. 8.69). A decrease in feed effi- ciency on acetic and an increase on lactic acid were noted for both nitrogen supplements (Table 22). The increased feed efficiency with added lactic acid is in agreement with trends observed in other work at the Michigan Station in which feed efficiency has increased with the level of lactic acid in the silage (Henderson gp_gl., 1971a; Cash, 1972; and Emery §E_§1., 1961). In addition, the ammonium lactate ration was the most efficiently utilized in Experiment I and one of the more efficient rations in Experiment II. Relative Value: The relative values of the acid additions to the two protein supplements are shown in Tables 21 and 22 and the derivation of this figure is discussed in Experiment I. Acetic acid additions reduced the value of the supplements but lactic acid additions had little effect on the value. Adding acetic acid and lactic acid to feedlot rations will probably never become an economic consideration, but these data do show that neither acetic nor lactic acid will reduce on con- sumption or impair feed efficiency when fed at the levels used in this experiment. 104 Carcass Evaluation: Lactic acid tended to slightly depress carcass grade in this trial. The group receiving lactic acid graded low Choice, whereas the control group and the group receiving acetic acid graded middle Choice. The groups receiving lactic acid had significantly (P < .05) less kidney, heart and pelvic fat than the other groups. Although the difference was not significant (P < .05), the group receiving lactic acid had only 1.37 cm. fat, whereas the control group and the group receiving acetic acid both had 1.52 cm. of fat over the rib eye. Soypean Meal vs. Urea: Data have been pooled for each nitrogen supplement and are shown in Table 23. Compared to cattle on soybean meal, those on urea gained 4.3 per cent less (804 9. vs. 840 9.), ate 2.6 per cent less DM (7.08 vs. 7.26) and were 1.9 per cent less efficient (8.80 vs. 8.64). Fat thickness of the group receiving urea was significantly less (P < .05) than for the soybean meal group (1.30 cm. vs. 1.65 cm.). Although differences in the remaining carcass traits were not significant, marbling scores and carcass grade values were slightly higher for the soy cattle. 105 Table 23.--Experiment III: Soybean meal vs. urea supple- ments (November 3, 1970 to April 22, 1971). Soybean Meal Urea 170 Day Test Supplement Supplement No. of yearling steers 17 17 Av. initial wt., kg. 298.3 299.6 Av. final wt., kg. 441.7 436.7 Av. daily gain, kg. .840 .804 Daily Feed, kg. 85% DM: Corn silage 1.71 1.66 Gr. shelled corn 5.03 4.88 Nitrogen supplement 0.52 0.54 Total 7.26 7.08 Feed Efficiency: Feed per kg. gain, kg. 8.64 8.80 Feed and yardage cost per 100 kg. gain 1 49.81 49.81 Relative value of supplements 91.86 84.46 Carcass Evaluation: Carcass grade‘ 13.15 12.79 Marbling score3 14.86 14.06 Fat thickness, cm. 1.65a 1.30 Rib eye area, cm. 4 69.93 68.77 Per cent K. H. P. fat 5 3.77 3.83 Per cent B. T. R. cuts 48.14 48.94 Dressing per cent6 61.82 60.76 Carcass price per 100 kg. $113.74 $114.80 1Feed costs based on 35% DM corn silage $9.37/mt., shelled corn $49.60/mt., yardage at lO¢/steer/day. 2Good = 9, 10, 11; Choice = 12, 13, 14. 3Small = 10, 11, 12; Modest = 13, 14, 15; Moderate = 16, 17, 18. 4Per cent of carcass weight in kidney, heart and pelvic fat. 5Per cent of carcass weight in boneless, trimmed retail cuts. 6Cold carcass weight over off experiment weight. Significance: Values having different superscript differ significantly; a (P < .05). 106 Experiment IV--Acetic and Lactic Acid Additions to All Silage Rations Acetic Acid Addition: The results of this trial are shown in Table 24. At all levels of addition, acetic acid depressed intake, gains and feed efficiency when compared to the control ration. However, none of these differences were significant (P < .05). The depression in DM consumption was intermediate (3.2 per cent) at the 1.5 per cent level of acetic acid addition and maximized at 3 per cent which was 8.4 per cent below controls. Higher levels of acetic acid did not decrease daily consumption below that observed at 3 per cent. These findings contradict the results of Experiment III in which acetic acid had no effect on daily consumption of a high concentrate diet. But they support the numerous reports of depressed DM intake with rumen infusions of acetate (Simpkins g£_§1., 1965; Weston, 1966; Rook etggl,, 1963; Baile and Pfander, 1965; and Montgomery gE_gl., 1963) and with higher levels of acetate in the ration (Dinius gp_31., 1968 and Wilkins gE_§l., 1971). Gains were decreased by 10.3 per cent, 15.2 per cent, 11.7 per cent and 7.2 per cent below the control steers at the increasing levels of acetic acid addition. This response is also different from that obtained with 107 O0.0 O0.0 N0.0 NN.O NO.H coHumssmcoo so mo memo nmm .hth mm.o mm.o mm.o mn.b Hmuoa OH.O OH.O OH.O HH.O NH.O momHHm 2H OH66 oHomom OO.O ON.O OH.O OH.O moon oHom onmom omoo< u.ax .oHom mHumms mo mxmusH mHHmo NO.O ON.O NO.O N0.0 HO.O .OH .chm .OH nmo omsomnoo so OO.N OO.H NO.H mO.N OO.N .oz soon so unmo nmn O0.0 NO.m OO.O mm.m ON.O Hmuoe ON.O ON.O OH.O OH.O moo: OHom oHomom .mwwm mwhm ON.O OH.O OO.O Hmooe O0.0 N0.0 O0.0 N0.0 OO.O unmsmHomsm HmnmcHsnsom mN.O NO.O OO.O OO.O OO.O momHHm nnoo ".ax .sOHmeSmsou Hmuumz who OO.O OO.O OO.O HN.O HO.H .OH .chO NHHmo .>m 0.0mm m.OOm 0.0mm N.Nom m.ONm .OH ..o3 Hmon .>¢ O.NON N.OON O.OON N.OmN O.OON .OH ..H3 HmHanH .>m OH OH OH OH OH mmeHmo nmmun mo .02 0.0 m.O O.m m.H Honmooo So maMHHm mo w .oHom 0Humom omoos Home smo ONH .AmbaH NMH SOHMS OH HhaH .HH Hmnfim>ozv msOHums mamHHm HHm on sOHuHoom oHom 0Hum0¢ ">H psmEHHmmetI.Om mHnme 108 the high concentrate ration and is probably a result of the decreased drymatter consumption. As in Experiment III, adding acetic acid to the ration decreased feed efficiency. Feed efficiency was decreased 8.6 per cent, 8.5 per cent, 5.6 per cent and 0.6 per cent by the increasing increments of added acetic acid. Lactic Acid Addition: Gains were not affected by adding lactic acid to the ration. There was also no difference in feed efficiency or DM consumption among the various levels of added lactic acid. The addition of lactic acid did result in a decreased consumption of the basal ration (silage and protein), but this decrease was offset by the lactic acid consumption. Similarly, Emery gE_§l. (1961) reported that lactic acid addition reduced appetite in proportion to its concentration when fed to growing heifers. These results differ from Experiment III in which lactic addition resulted in both an increased average daily gain and feed efficiency. Emery gE_§1. (1961) reported that feed efficiency increased in propor- tion to lactic acid intake. 109 OO.mH mO.NH m0.0 NH.N O0.0 oOHudssmcoo so mo Homo smo O0.0 HO.O N0.0 NO.O OO.O Hmooe NN.O NN.O ON.O ON.O ON.O mmmHHm oH oHom onomH O0.0 OO.O OO.O NH.O mnon oHom oHoomH omoom "max .oHod UHuomH mo mxmusHONHHmo Hm.O OO.O NN.O HN.O HO.O .OH .nHmO .OH nmm omsoncoo 2O NH.N OH.N OH.N OH.N OO.N .oz Noon mo Homo nmn mm.O OO.O O0.0 O0.0 ON.O Hmuoe OO.O OO.O ON.O NH.O mnon OHom oHnomH NO.O OO.O ON.O mm.m O0.0 Hmuoe NO.H OO.H N0.0 O0.0 O0.0 ucmsmHmmsm HmsmsHsuNom OO.O OO.O NO.O OO.O OO.O mOmHHm anoo ".ax .sOHAmESmsOU Hmuumz hum NO.H HO.H OO.H NO.O HO.H .OH .eHmO NHHmo .>m 0.0NO m.Nom 0.0NN N.OOO 0.0NO .OH ..H3 Hmch .>m N.OON O.OON O.OON N.NON O.OON .OH ..03 HmHoHoH .>< OH OH OH OH OH nm>Hmo nmmun mo .02 O.OH m.N O.m m.N Honmooo So maMHHm no N .onm UHuUMH omoos Home Nmo ONH .HmhaH .MH Sumo: 0» HbaH .HH HmQEm>ozv msoHums mamHHm HHm on sOHuHoom onm OHuomq ">H usmEHHmmxmi..mm mHQmB 110 Ex eriment V--Study of Rumen ang Blood Parameters Associated with the Addition of Ammonium Salts or Organic Acids—to Cattle Rations Rumen Ammonia and Blood Urea Concentrations for Nitrogen Sources: Mean values for rumen ammonia and blood urea for the various nitrogen sources are shown in Table 26. The rumen ammonia concentration was highest at T (2.5 hours post feeding) for urea, ammonium ace— 2.5 tate and ammonium lactate supplemented steers. The soy- bean meal fed steers had a maximum rumen ammonia concen- tration at T (immediately before being fed). Rumen 0 ammonia was significantly higher P < .01) for the cattle fed ammonium salts at T than for the urea or soybean 2.5 meal fed cattle. Cattle fed ammonium lactate also had the highest (P < .05) rumen ammonia levels at T5 and T10. The blood urea levels were higher than urea or soy fed cattle at all sampling times for the cattle fed ammonium salts, but the differences were not significant and T (P < .05) at T At T5, blood urea levels were 0 2.5' significantly greater for cattle fed ammonium lactate (P < .01) or ammonium acetate (P < .05) than for cattle fed urea or soybean meal. None of the differences in blood urea between ammonium acetate and lactate fed cattle or between urea and soybean meal fed cattle were significant (Table 26). 111 .HHO. v mo m .< uHmo. v av .Q.m "mHusmoHMHsaHm HmMMHo mumHsomsmmsm usmHmMMHo asH>ms mmsHm> .mfimmg MO HO.H.HG UHMUGMflm " mm N .smmE Hmm msOHum>Hmmno meH OO.O OO.HH NO.O NN.N ON.O OHe on non mm on . . . . O m OO O omNH NH oomO OH mommO O momNO O s HO.O ON.HH NO.HH NO.O OO.O O Na OO.O ON.O NO.O OO.O NO.O Oe mmHD ooon OO.O ON.N OH.H ON.H OO.H OHe fl mm m m . . . . . m OH O 9HON H mNH H mOO O mmNN O O . . . . . m.N OO H mNO OH mON OH mom O «OO O a O0.0 OO.N NN.N OH.N NN.O Oe MHsoafis smesm Nmm mummomH Omz mumomo< Omz mmso Now mEHB monsom smaosqu .HHE OOH\an muHmm EsHsOEEm omm mummum How mmsHm> mmss oOOHn osm MHsOEEm smEsH Hsmmz u> usmEHHmmxmll.om mHnme 112. Rumen VFA Concentrations for Nitrogen Sources: Rumen acetate, propionate and butyrate levels for cattle supplemented with nitrogen from various sources are shown in Table 27. Rumen acetate concentration was higher at all determinations for cattle fed ammonium lactate than those fed ammonium acetate or urea. The difference was highly significant (P < .01) between ammonium lactate and urea at T0 and significant (P < .05) between ammonium lactate and soybean meal or ammonium acetate at T0' Cattle fed both ammonium acetate and ammonium lactate had higher rumen acetate levels at T than soybean 2.5 meal. Differences in rumen propionate levels were not significant (P < .05) at T , or T However, 0' T2.5 10' ammonium lactate and soybean meal supplemented cattle tended to have higher rumen propionate concentrations at all post feeding determinations. Rumen propionate concentrations were significantly higher (P < .01) at T2.5 for the ammonium lactate cattle than for any other group. Urea fed cattle had the lowest (P < .05) rumen propionate level at T2.5. Rumen butyrate was not detectable in rumen fluid samples of the ammonium lactate fed steers at any sampl- ing time. The cattle fed urea had significantly lower and T than 0 2.5 cattle receiving soybean meal or ammonium acetate at T10- (P < .05) rumen butyrate levels at T 113 .HHO. “30. v mo .Q.m umHusmoHMHsaHm HmMMHo mpmHHomHmmsm usmHmMMHo asH>ss mmsHm> .msmmS mo Hosnm osmosmum u mmN .smmE smm msOHum>smeo meH OO.O O NO OO OO OHN U am n4 Md m NO o mo «mo sNO smm m.NB mO m mo odoo nsmv msoo o9 mm M De mmmv nsNN msmm B mumsmwsm smEsm NH.OH OO Om OO OO OHH OO.OH OHH OO NN NO ON . m.N oN HH mNoH QOoHH MOMN noHHHmHH o9 Nm.oH NO No NO ON 9 mumsonaHm smfism ON.ON NNN NOH OOH NON OHH NO.NO NOO NON NON NON me . m.N mm MO omOmO oonm mmmN onmmN o9 om NH soHEN omoUmNN oummNH ooooHN a mumumos smssm mm mummomq Omz mumumom Omz mmno Now N mEHB moHSOm smaosqu .muHmm SHGOEEM UQM mhmmfim HOW mCOH#MHHGGOGOU fig G05“. H smmz n> usmEHsmmeIl.NN mHQme vmvm£ 114 § g. ‘ is. Rumen butyrate levels were significantly higher (P < :05) for cattle fed soybean meal than for those receiving either ammonium acetate or urea. Rumen Ammonia and Blood Urea Concentrations of Acid Fed Cattle: The differences in rumen ammonia levels (Table 28) were not significant at T or T 0 2.5' rumen ammonia was higher at T2 5 for the cattle fed lactic However, acid ration. At T the lactic acid fed cattle had 5: greater (P < .05) levels of rumen ammonia than the control cattle. The rumen ammonia concentration for the lactic acid fed cattle was significantly higher at T10 than for cattle on control (P < .01) or acetic acid (P < .05) rations. Blood urea levels were highest at all sampling times for lactic acid fed cattle, especially at T5 when blood urea was significantly higher than for cattle receiving the control (P < .05) or the acetic acid ration (P < .01). The blood urea levels for the cattle on the acetate ration were significantly lower (P < .05) than the lactic acid fed cattle at all post feeding sampling times. Rumen VFA Concentrations for Acid Fed Cattle: Rumen acetate concentrations were higher (P < .01) at T0 for the lactic acid fed cattle. All other differences in rumen acetate concentrations were not significant, but the acetic fed cattle had the highest concentrations 115 .HHO. v my m.< “Hmo.uvmv n.m ”mHusmonHsaHm HmNMHo mpmHHomstSm usmHmMMHo asH>ms mmsHm> .HH xHosmmms sH mum mmsoua oHUMIsHmuOHm HmsoH>HosH ms» mom msmmzm .mfimwe HO HOHHT UHMUCMflm H WWN OGMQE Hmm mGOHUM>H0mQO m>H®3B H OO.O OO.O OO.O OO.N OHN oom mm ommm O OO O omOO O OHOO N OmHOO O O.Ne NO O oNO OH mON O omOO O OH NO.O OO.O OO.N OO.O a "mmHD ooon HO.O ON.O OO.N NO.H OHN non MOO 6O O OO O oHO N omOO H mOO O O.Ne OO.O OO.O ON.O ON.O e O0.0 OO.N ON.N ON.O Os "EMHsOEfid smEsm mm oHom OHHOMH oHom 0Humu< Hosusoo N mEHB omusmEmHmmsm oHos .H.HE ooH\.aEv mummum omm oHom How mmsHm> mmss oooHn osm EsHsmEEm smesu Hsmmz .> usmEHHmmxm:..mN mHQMB 116 at T and the lactic acid cattle had the highest con- 2.5 centration at T5 and T (Table 29). 10 Rumen propionate concentration at T2.5 was significantly higher (P < .01) for cattle fed lactic acid than either the control or acetate fed groups and higher (P < .01) for cattle fed lactic acid than for those fed acetic acid at T The acetic acid fed cattle 5. had the lowest rumen propionate concentration at all sampling times, and they were significantly lower (P< .05) than the control group at T5. The control group had a significantly higher rumen butyrate level than the lactic acid fed steers at T2.5 (P < .05) and higher than the control or lactic acid group (P < .01) at T5 and T10. Rumen butyrate was not detectable for steers fed urea in combination with lactic acid, but when soy was fed in combination with lactic acid the rumen butyrate levels were higher than any other protein-acid combination at T0, T2.5 and T5. The means of the individual protein-acid groups are presented in Appendix II. Rumen Ammonia and Blood Urea Concentrations on Soybean Meal and Urea Rations: The results of this study are presented in Table 30. Rumen ammonia concen- tration was significantly higher at T for cattle fed 0 soybean than for those fed urea. Rumen ammonia concen- tration was maximized at T and was slightly higher 2.5 117 .HHO. “Hmo. v my n.m "mHusmmHMHsaHm HmMMHo mumHsmmummsm usmHmMMHo asH>ms mmsHm> .HH xHosmmm¢ sH mum mmsosa oHomusHmuosm HmsoH>HosH man How msmmz v mo m.< O .msmmE mo Housm osmosmum u mmN .smmE Hmm msOHum>ummno m>Hm39H . OH OO O mON mOO «NO ON NO O mOO mHO «OO O.Ne HN O oOO mHO mNO Oe NO.O ON OO ON a mumuNwsm smfism HN.N NO OO ON OHH . m NO O mmOO e.HHO mmONO O.Ne OO O mOOH «OO «OO ON ON.N ON HO OO O mumsonmHm smasm OO.OH NON HOH OHN OHN ON.OH HON HON NON ON OO.NH NHO ONO OON O Na . O OO OH mNOO OONN «OOH e mumumos smasm Nmm oHom mHuomq onm UHumos Hosusou omusmEmHQQSm oHU¢ mH.Hs OOH\.OsO mEHB mummum omm oHom How msOHumsusmosoo ¢m> smEsH smmz u> usmEHHmmxmll.aN mHQma 118 Table 30.-Experiment V: Mean1 blood and rumen values for urea and soybean meal supplemented animals (mg/100 ml.).3 Time Soy Urea SE2 Rumen Ammonia To 3.54a 1.93 0.43 T2.5 4.54 5.17 0.74 T5 1.78 1.52 0.29 T10 2.65 2.12 0.42 Blood Urea To 7.01 8.01 0.46 T2.5 8.64a 10.21 0.43 T5 8.13a 9.33 0.33 T10 7.58 8.54 0.40 Rumen Acetate To 240 242 10.97 T2.5 302 299 14.58 T5 246 270 15.27 T10 216 224 15.84 Rumen Propionate To 73a 56 5.88 TZOS 122 100 7.90 T5 83 79 5.61 T10 70 61 5.89 Rumen Butyrate To 39A 17 3.12 T2.5 63A 31 3.03 T5 49A 28 3.65 T10 49A 26 3.66 1 2 3Means for the individual protein-acid groups are in Appendix II. Values having different superscripts differ signi— ficantly: a (P < .05); A (P < .01). Eighteen observations per mean. SE = Standard error of mean. 119 for the urea cattle than the soybean meal cattle at this time. Blood urea levels were significantly higher 2 5 and T5 for the urea fed cattle. Blood urea was maximized at T2 5 for cattle on both supplements. Rumen VFA Concentrations on Soybean Meal and Urea (P < .05) at T Rations: There was practically no difference in rumen levels of acetic acid between cattle receiving the urea or soybean supplemented rations. Rumen acetate levels were highest at T2.5 for both nitrogen sources. Propionic acid levels in the rumen were highest at all determinations for the soy fed cattle, and the difference was significant at T The differences at 0. all other sampling times were not significant. Rumen butyrate levels were significantly higher (P < .01) at all sampling times for the soybean meal fed steers. Experiment VI--Nitrogen Balance Study wipp Ammonium Acetate, Emmonium Lactate, Urea and Soybean Meal Rumen Ammonia and Blood Urea Concentrations: Com- parison of rumen ammonia and blood urea levels are shown in Table 31. None of the differences in these parameters were significant. Rumen ammonia levels were maximized at T2 for all rations. In contrast to Experiment V, the rumen ammonia :. _ __ _- _ _.......-r.l .msmmE smmzumn mmosmHmMMHo usMOHMHsaHm oz .smmE mo Hosnm osmosmum H mm 120 N .msOHum>Hmmno Hsom mo msmmE mmumsam ummmHH . . C C C CH ON H OO O OO N OO OH OO O a NO.H O0.0 OO.O OO.NH O0.0 ON OO.H OO.O ON.O OO.NH OO.O ON OH.H OO.O OH.O ON.NH OO.O Oe OO.O ON.N ON.O OO.OH OO.O Ne OO.O OO.N OO.N OH.O OO.N Oe mmHD ooon OO.O OO.O ON.O ON.O ON.O OHH HO.N ON.O OO.H OO.O OO.O Oe OO.O OO.O OO.N O0.0 OO.O Oe OO.O OO.OH OO.O OO.OH OO.O ON OO.N O0.0 OO.O OO.O OO.O Ne OH.H OO.O OO.O OO.N ON.O Oe mHsOEfis smfimm Nam momuomH Omz mumumo< Omz mmso Now usmEmHmmsm smaosqu .H.HE ooH\.aEV mmsHm> mmHs oooHQ osm MHsOEEm smEsn mEHB OH> usmEHHmmxm:l.Hm mHnms 121 level for the urea fed cattle was higher than for cattle fed ammonium salts at all sampling times except T10. The blood urea levels of the urea fed cattle were also higher than for cattle fed the ammonium salt or soybean meal supplemented rations. Varner and Woods (1971) reported that rumen ammonia levels were higher at one hour post feeding for cattle fed ammonium salts than for urea. Serum urea levels were highest for urea fed steers, intermediate for steers fed ammonium salts and lowest for soybean meal fed animals. In a second study, reported in the same paper, they found that serum urea levels were higher for the ammonium salt supplemented steers than for urea fed steers. Rumen VFA Concentrations: Table 32 shows mean values for acetate, propionate and butyrate concentra- tions expressed as mg. of VFA for 100 ml. of rumen fluid. Rumen acetate concentrations were highest for the ammonium lactate fed cattle at T0 and T2. Although these differences were not significant, they do agree with the results in experiment V. Rumen acetate levels reflected the addition of ammonium acetate to the ration. The probable explanation for increased rumen acetate levels when lactic acid and ammonium lactate are fed is the catabolism of lactic acid to acetic acid. Jayasuria and Hungate (1959) and Bruno and Moore (1962) reported 122 Table 32.--Experiment VI: Mean1 rumen VFA concentrations (mg./100 ml.). 1 1 -:‘ Nitrogen source Time NH4 NH4 2 S°Y urea Acetate Lactate SE Rumen Acetate T0 303 322 341 439 61.86 T2 288 312 325 331 67.35 T4 355 300 392 394 63.64 T6 360 317 412 399 50.44 T8 389 347 351 370 64.08 Tlo 387 322 362 381 30.97 Rumen Propionate T0 159 234 130 198 36.43 T2 141 142 120 189 33.41 T4 116 205 141 205 43.58 T6 166 156 146 212 44.40 T8 172 236 133 205 46.73 T10 202 152 187 202 30.74 Rumen Butyrate T0 39 78 141 145 51.17 T2 42 119 118 150 43.67 T4 63 56 105 159 23.14 T6 56 60 140 125 29.23 T8 57 64 116 126 30.42 T10 61 90 91 117 34.85 1Least squares means of four observations. 28E = Standard error of mean. No significant differences between means. 123 that acetate was the major conversion product of lactic acid in the rumen. Rumen propionate levels were also increased by supplementing the ration with ammonium lactate. Ammonium acetate fed steers had the lowest rumen propionate con- centration at all sampling times except T4. At T4, the rumen propionate level of acetate fed steers was lower than for steers fed urea or ammonium lactate but higher than on soybean meal. Rumen butyrate concentrations were higher for steers fed ammonium salts than for steers fed urea or soybean meal. The urea fed steers did have levels of rumen butyrate at T2 and T that were comparable to the 10 concentrations found in ammonium acetate fed steers at the same time. Dry Matter Intake and Digestibility: As shown in Table 33, the ammonium acetate fed cattle consumed less dry matter daily than any other treatment group. The reduction in dry matter consumption for the ammonium acetate fed steers was 12.0 per cent, 10.9 per cent and 17.2 per cent when compared to the soybean meal, urea and ammonium lactate fed steers, respectively. These differences were not significant. The ammonium lactate supplemented rations had the greatest dry matter digestibility (72.6 per cent) and was followed by ammonium acetate (69.7 per cent), 124 .Aao. v my m.¢ "maucmoflMHsmHm HGMMHG mumwuomummsm ucmHmMMHU mca>mz mmDHm> .mcmmE mo Houum oumpcmum u mmN .mcmma mumsvm ummmq H mm.> mm.h mm.¢ «a.mml «m.mml mxmucfl 2 mo m mm Umcfimumu .z em.e ma.ma mm.e «a.mmu e~.emu mme\.a .tmaamumu emaoupaz mm.m m.ee a.me a.me m.em mmo\.m .emmouuac mumcaup em.m a.mm m.em a.me a.me a .emummmae amaouuae uemo uma mm.mH m.ea m.mm m.ae e.om ame\.m .emummmaa ammonuaz mo.m e.Hm a.mm a.mm H.em ame\.m .emmouuae Hmomm em.ma m.eee a.mma m.moa H.0HH mma\.m .mxmuea ammonpaz em.~ a.mn a.mm e.em a.em w .emummmae so ucmo awe m.HHm omom mace came move amo\a .emummmae 2e m.aam ammo seem ammo meet ama\a .mxmuea 26 II a v v v mummum mo .oz Nmm muwwumq muwmmofi mono wow mousom somouuflz H .mumumfimumm coflummmwp so mmOHSOm cmmonuflc mo muowmmm nH> quEHHmmeII.mm magma 125 soybean meal (67.9 per cent) and urea supplemented rations (64.6 per cent). The digestibility of the urea supple- mented ration was decreased by 4.9 per cent, 7.3 per cent and 11.0 per cent when compared to soybean meal, ammonium acetate and ammonium lactate supplemented rations, respec- tively. Nitrogen Balance: The mean values for all nitro— gen balance parameters are shown in Table 33. The differ— ences in nitrogen intake, fecal nitrogen, urinary nitrogen and nitrogen digestibility were not statistically signifi- cant (P < .05). Steers fed ammonium salts had greater nitrogen intakes but did not lose any more nitrogen in the feces or urine than the soybean meal and urea fed steers. This resulted in an increased quantity of nitrogen digested and an increased nitrogen retention. The urea and soybean meal supplemented steers were in a negative nitrogen balance and retained significantly (P < .01) less nitrogen than ammonium acetate or ammonium lactate fed steers. Experiment VII-—Nitrogen Balance Study Comparing Various Ammonium Salts of Organic Acids with Con— ventional Nitrogen Supplements Rumen Ammonia Concentrations: Tables 34 and 35 show the data from the study of rumen ammonia and blood urea levels in this experiment. 126 V :iuhs. 1.5.... ...e _. . I .Amo. v mv o.n.m “maucmoflwflcmflm mommap mumwuomnmmsm ucmnmwmwp maw>ms mosam> .mcmmfi mo Hounm pumpcmum H mm m .cmmE Mom mGOHum>Hmeo mounea . C . . . . ' C . OH mo H m m o m m m a m o m e m o a e a e em.a o.m o.m o.m m.m o.m o.e e.m e.m we ae.o o.m o.e m.m e.e a.e m.m e.~ e.m we HH.H e.m 0.4 m.m e.e o.o 5.4 o.m m.m we . C C O . . . C . N we m om NH am mm ohm ea ham mm onmo ea one ma ohm ea oo NH 9 £4 m.m mg me Be e6 m.m 5m 04. ee mumumusm mumuomq mumcoflmoum mumumod mumEnmm mum emz vmz emz emz emz ammo emu a mom mafia mUHSOm cmmouuwz .A.HE ooa\.mEv mcowumuucmonoo wwGOEEm Goes“ ammo: "HH> ucmEHHmmxmll.vm manna 127 .mcmoa cmmzumn moocmnmmwflp unmoflmwcmwm oz .m:0aum>ummno Gonna no name mnmavm ummmq .smmE mo Hounm pumpcmum fl mmN H ao.H ~.m m.a a.m a.m m.oa o.m a.m a.a oee ma.e m.a ~.a ~.a m.eH ~.oa e.m a.m e.m me ao.a m.oa H.m H.eH m.HH a.ma e.m N.OH e.oa we m~.H m.HH e.a o.HH a.ma a.ma m.m m.oa a.eH we mo.H a.oa m.HH o.HH m.ma m.mH H.0H a.HH N.HH me we.o a.m m.m a.m a.a m.m «.5 e.m a.m oe mum wuwmmusm ouwwqu oumcwwmonm muwmmod muwmmmm mono 3mUID mom mEHB mUHSOm cmmouuflz H .A.HE ooa\.mEV mGOHHMHmeonoo nous COOHQ com: "HH> usmEHHmQxMII.mm magma 0128 As in experiments V and VI, rumen ammonia values were maximized at T2. The ammonium lactate fed steers had significantly (P < .05) higher rumen ammonia values at T2 than steers fed ammonium propionate, U-CSW, Urea, ammonium butyrate or soybean meal supplemented rations. Cattle fed ammonium acetate had significantly (P < .05) higher rumen ammonia levels than those fed ammonium butyrate or soybean meal. All other differences in rumen ammonia levels were not significant. The relative rumen ammonia levels for ammonium acetate, ammonium lactate, urea and soybean meal fed steers paralleled the results of experiment V but do not agree with the results of experiment VI. The results of experi- ments V and VII agree with previously published work (Varner and Woods, 1971). There was virtually no difference in rumen ammonia concentration, among the various nitrogen sources, other than the difference at T . 2 Blood Urea Concentrations: There was no signifi— cant difference in blood urea levels among the cattle fed the various nitrogen sources tested (Table 35). Maximum blood urea levels occurred between T2 and T6 for all treat- ments. Steers fed ammonium acetate and ammonium formate had the highest blood urea levels. The relative blood urea level of the urea fed steers was lower than expected. 129 Blood analysis in experiment VI showed a higher urea level for the urea fed steers than for soybean meal or ammonium salt fed steers, but the same analysis in experiment V showed higher blood urea levels for the ammonium salts. Varner and Woods (1971) reported higher blood urea levels for urea fed steers when compared to ammonium salt fed steers in one trial and higher blood urea levels for ammonium salt fed steers in another trial. _ _--._.-.-fl Rumen Acetate Concentrations: Rumen acetate con- centrations (Table 36) were highest for ammonium acetate fed steers at T2 but significantly (P < .05) lower for the ammonium acetate fed steers than for steers supple- mented with soybean meal at T This same trend was 4. observed in experiments V and VI and for the addition of both ammonium acetate and acetic acid to the ration. The rumen acetate levels were elevated in steers fed rations supplemented with soybean meal or ammonium lactate. These same trends existed in experiments V and VI. Soy fed cattle had significantly (P < .05) higher rumen acetate concentrations than cattle fed rations supplemented with urea, ammonium acetate or ammonium propionate at T U-CSW fed cattle had significantly 4. (P < .05) higher levels of rumen acetate than urea or ammonium propionate fed cattle. All other differences in rumen acetate levels were not significant (P < .05). 130 .Amo. v my 0.3.6 “mausmofimwcmflm mommap mumflnomummam ucmummmap mcw>mn monam> .mcmmE m0 Honum Unmpcmum u mmm .Gmme mom mcowum>uwmno mouse H mH.mm «mm mme aHm emm cam mem mme wee 0H9 mo.ae Hem ham oom hem omm Hmm hem ewe we mm.em mam mmm mmm mam emm 5mm mam Ham we . e ee me oanoe oammee oeHm unmem unmome comm ammme mmem e ma.~m cam mme emm nae omm mme mam Nee Ne em.me emm eme Ham emm ham mme mem OHm oe mumuhusm mumuomq mumsowmonm wumumod mumfiumm mum emz emz emz emz emz ammo smoo mom msHe moonsom cmmouuflz .A.HE ooa\.mEv mGOHumuwsmocoo mumnmom cmfisu dado: "HH> unwewnmmxmll.mm magma 131 Rumen Propionate Concentrations: The rumen pro- pionate levels of steers fed various nitrogen sources are shown in Table 37. Supplementing the ration with soybean meal or ammonium lactate resulted in elevated rumen pro— pionate levels. The rumen propionate concentration was higher for ammonium propionate supplemented steers at T2 than for steers receiving other supplements except ammo- nium lactate. The difference in rumen propionate concentration between ammonium lactate and ammonium propionate was not significant at T Steers fed ammonium salts of lactic 2. or propionic acids had higher (P < .05) rumen propionate levels at T2 than steers fed rations supplemented with ammonium formate or ammonium butyrate. The ammonium lactate fed cattle also had significantly (P < .05) higher rumen propionate levels than cattle fed soybeam meal, urea, U-CSW and ammonium acetate. At T4, rumen propionate levels were significantly higher (P < .01) than urea or ammonium acetate fed cattle and higher (P < .05) than for cattle fed ammonium buty- rate or ammonium propionate. Soybean meal fed cattle had higher (P < .05) rumen propionate levels than steers receiving urea, ammonium butyrate or ammonium acetate. The difference between the U-CSW fed cattle in rumen pro- pionate at T and cattle fed urea or ammonium acetate was 4 also significant. 132 .Amo. v mv o.n.m “maucmowmwcmflm HmMMflo mumfinomHGQSm .mcmmE .GMQE .AHo. v mo o.m.m ucmHmMMflp mcw>ms mo5am> MO HOHHU UHMUCMHM N HMN mom msoflum>ummno wmnsaa OH.aH ma aHH H» me am aOH mHH NHH OHe mm.e~ mo mHH an MOH am mm mm ooH we om.eH MOH MHH em aOH eOH eeH as meH we . e Ha mH HvooemmOH memeH.eoeemmHH emoa emememamH comem onmmemmH ememmmH e . N on mm omeHH mmomm emmemmm Hue.eeeH oeomH 0eeeeH ohmmeH OOHeHeH e HO.ON om NMH Ha em ma FOH mm mm as mumnmusm mumuomq mumcowmonm mumumo< mumeumm mum emz emz emz emz emz «was emu: mom wsHe mooHSOm cmmouufiz .A.HE ooa\.mEV mcoflumuucmocoo mumcoflmoum smash ammo: "HH> ucwfiwummxmll.hm magma 133 The data from this experiment combined with results from experiments V and VI suggest that a major intermediate in lactic acid metabolism in the rumen is propionic acid. This agrees with conclusions of other workers (Elsden, 1945; Philipson, 1952; Waldo and Schultz, 1956; Montgomery §£_El,, 1963). Supplementing rations with soybean meal resulted in higher rumen propionate levels than feeding the other nitrogen sources except for ammonium propionate at T2 and ammonium lactate at all sampling times. This may have been a result of a stimulation of propionic acid fermentation or a decrease in the inhibition of propionic fermenters as compared to the other sources of nitrogen. Bumen Butyrate Concentrations: Rumen butyrate was highest at T T and T on the ammonium butyrate 2' 4 6 supplemented ration (Table 38). When acetate or pro— pionate was added to the diet, the rumen concentration of the respective acid appears to be maximized at T2 and decreases rapidly thereafter (Experiment V, VI and VII). This may be a result of an increased rate of acetate or propionic metabolism as absorption when these acids are added to the diet. Rumen butyrate at T4 was higher (P < .05) for ammonium butyrate fed cattle than for those fed other rations and higher (P < .05) for cattle fed ammonium butyrate, soybean meal and ammonium lactate than for 134 .xHo. v we o.m.¢ aSo. v my o.n.m “mauGMUAMHcmam umMMflU mumwuomHmQSm uanmMMflo msfl>m£ mmsHm> .mCMGE MO HO.H.HQ UHMUGMHW H HMN .cme mom mGOflum>uomno donned mm.o~ eOH eeH mm mm mmH we mHH HmH 0H9 mm.m~ em oeH mOH eHH ONH em km HaH we mm.~m eaH mOH mm omH OMH moH we meH we me.mm memHm mmeeH HvomHa UunmoS oatmemH amen emmmmH memem ee em.m~ memem onemeH omam UQOMH 0emHH oanH uanH enemaH Ne em.em am mmH ma eOH eeH meH me em 09 mGOHSOm cmmouuflz .A.HE ooH\.mEV mCOAuMHucmocoo muMHMpsn cmfisu Hammz .HH> unmeflnmmxm.:umm manna 135 those fed urea or ammonium salts of acetic or propionic acid. Urea fed cattle had the lowest (P < .05) rumen butyrate level at T The increased level of rumen 4. butyrate with lactate supplementation is in agreement with work by Montgomery gt_gl. (1963) who reported in- creased rumen butyrate concentration with lactic acid administration. However, these results disagree with those in Experiment V in which a combination of lactic acid and NPN resulted in an unexplained absence of rumen butyrate. Rumen pH: Differences in rumen pH were small and insignificant at all times of sampling except T4 (Table 39). Rumen pH was lowest on the soybean meal supplemented ration except T and rumen pH was signifi- or cantly (P < .05) lower on soybean than other rations at T4. Rumen pH at T was higher (P < .05) for the ammo- 4 nium propionate ration than for the U-CSW, ammonium lac- tate or soybean meal ration. Drngatter Intake and Digestibility: None of the differences in dry matter intake or digestibility were significant (Table 40). Soybean meal fed steers had the lowest dry matter consumption and the least dry matter digested. Nitrogen Balance: There were no significant dif- ferences (P < .05) in any of the nitrogen paramers (Table 40). 136 .HHo. v my o.m.m .Amo. v mv 0.9.m uhHucmoHMHcmHm HmMMHo mumHHomHmmsm ucmumMMHU mcH>mn mmsHm> .mcmme mo Honnm Unmocmum u mmm .cmofi Ham mcoHum>Hmeo ooHSBH eHH.o mm.e He.m mm.e ea.o 05.0 mm.m mm.m em.m eHe «HH.O mm.m mm.m no.n em.o em.m Hm.m m>.m hm.m ma mNH.o o>.m ~>.m m>.m n>.o om.m mn.o mm.m em.m we 0 O O O O O O O C v moo o QmUmh m Gnomow m mmmm m QMUmw w Unmowm m nwoNh m onU mm cmEnn com: .HH> ucmEHHmmxmll.mm mHnma H 137 .mcmma cmmzuon moocmumMMHn Hmo. v my HGMOHMHcmHm oz .msmma mo Honum pumpcmum u mm N .mnmos mumnvm unmoHH eH.o a.mH m.m «.OH m.e S.MH m.OH e.H~ o.mn mxmueH 2 mo H mm emchuou z ee.m a.mH a.mH m.eH m.e a.mH e.mH m.m~ a.ms ame\.m .omeHmumu ammouqu mm.m o.em o.oo «.mo m.Ho m.mm H.eo a.mm m.em ame\.m .emmouuH: mumaHus me.m m.oe m.mo e.oa a.mo m.oe m.oo m.oe 5.00 a .emummmHe emmouuH: memo awe mH.0H a.me H.~e H.Hm m.mo m.ea m.oe m.mm m.Hm ame\.m .nmummoHe nomouqu o~.m a.mm e.oe a.mm m.Hm m.Hm e.mm m.om m.em ame\.m .emmouuH: Hmomm ON.NH w.~0H a.mHH m.MHH m.OOH m.oOH m.eHH o.omH H.mm amt\.o .mxmucH comouqu mm.H a.me m.ee H.5e e.me m.ee e.me e.oe H.me e .emumomHa an Heme uma e.emm enmm meme emHe ommm mmem mHmm emme omen amc\.m .amummmHe an a.mee meme mmom emmm omae mHme eHmm swam Hmme amc\.m mxmuaH an .n m m m m m m m m mummum mo .02 Nmm oumwmwsm mumwmma mumswwmoum wuwwmod muwmnzmm mono Swot: how mou50m :mmouqu H .mumumEmnmm COHummmHo so mmoHDOm cmmOHUHc mo uommmm "HH> ucmEHHmmeIl.ov mHan 138 All nitrogen sources except soybean meal promoted positive nitrogen balances. Daily nitrogen intake was highest for the U—CSW fed steers, lowest for the soybean meal fed steers and intermediate for cattle fed urea or ammonium salts. As in Experiment VI, urine and fecal nitrogen losses were relatively constant and the decreased nitrogen intake on the soy ration was probably the major cause of the nega- tive balance. Daily nitrogen retention was 29.8 g, 15.9 g, 15.0 g, 14.9 g, 12.4 g, 12.0 g, 7.8 g and -3.0 g for the U-CSW, ammonium butyrate, ammonium formate, ammonium propionate, urea, ammonium lactate, ammonium acetate and soybean meal fed cattle respectively. As in the compara- tive feeding trial (Experiment II) ammonium acetate was the poorest ammonium salt tested. GENERAL DISCUSSION Growth studies with cattle in this dissertation and in other work (Varner and Woods, 1968; 1969) have consistently resulted in gains that were greater for cattle fed ammonium salts than those fed urea. In fact, gains of ammonium salt fed cattle are frequently equal to or superior to those of cattle fed soybean meal. Feeding ammonium salts did not affect DM con- sumption in these experiments. The relative commercial value of the ammonium salts was 2 to 3 times greater than for urea and comparable to soybean meal. Nitrogen balance and metabolic studies were conducted in an attempt to determine why ammonium salts were superior to urea. Digestibility of rations supple- mented with ammonium salts was slightly higher than for those supplemented with soybean meal or urea. In ad- dition, the nitrogen retention for steers fed ammonium salts was greater than or comparable to those fed soybean meal or urea. Both ammonium lactate and ammonium acetate fed steers had significantly (P < .05) greater nitrogen re- tentions in Experiment VI but none of the differences 139 140 were significant in Experiment VII. Soy fed steers were in negative nitrogen balance in both experiments. The variability in nitrogen retention is primarily a result of differences in nitrogen intake and not of nitrogen excretion. Steers in collection stalls were fed 90 per cent of their consumption during the acclimation period but many of the steers still went off feed during the collection period. Since there is a digestive lag between consumption and excretion, the collection cf excreta compares to consumption only when the consumption is uniform from two to three days prior to collection until the collection period is completed. The consistency of results should be improved if steers were acclimated to the collection stalls prior to collection. Rumen ammonia and blood urea were measured in experiments V, VI and VII. Feeding ammonium salts re- sulted in higher rumen ammonia levels than feeding urea or soybean meal at the first post-feeding sample when consumption was limited to the first two hours of feeding. Ammonium salts result in an immediate increase in rumen ammonia because the ammonia is immediately available, but the urea must be hydrolized before its ammonia is available. The results of Experiment VI are different from other data because steers consumed their diet throughout the day as usually occurs when steers are full fed in a feedlot. However, this makes comparison of metabolic 141 parameters more difficult with a small number of steers because of differences in consumption patterns. For this reason, the feed was removed from the steers two hours after feeding in all other metabolic studies. The highest concentrations of blood urea occurred at the same sampling time as the maximum rumen ammonia concentrations. If samples had been collected at shorter intervals after feeding, rumen ammonia should have peaked prior to blood urea. Blood urea was higher for ammonium salt fed steers than for those fed urea or soybean meal. By our current interpretation of this data, we would predict ammonium salts to be inferior to urea. But ammonium salts have consistently promoted greater and more efficient gains than urea. Another source of urea nitrogen in peripheral blood is nitrogen obtained from catabolism of body tissue and absorbed amino acids. If the steer was absorbing greater quantities of amino acids from the small intes- tine (as suggested by increased growth and nitrogen balance), the catabolic contribution to blood urea should be greater. To outline the metabolism of ammonium salts, more sophisticated measures are indicated. Analysis of digesta passing into the abomasum or small intestine would provide information concerning the synthesis of natural protein 142 and the loss of ammonia from the rumen via this pathway. Analysis of portal blood would be a more sensitive test for the absorbtion of ammonia from the gastrointestinal tract and would also provide information concerning amino acid absorption. Connulation of the rumen vein should be the most accurate method of determining the absorption of ammonia from the rumen into the blood. Rumen VFA concentration reflected the ammonium salt fed. Feeding ammonium salts of acetic propionic or butyric acids resulted in an increased rumen concentration at T for the respective acid. Rumen butyrate levels 2 were higher for steers fed ammonium butyrate through T 6' but feeding ammonium acetate or ammonium propionate re- sulted in an increased disappearance of the respective acids from the rumen. By T4, the concentration of these acids in the rumen was lower when they were fed than for the other nitrogen sources tested. Feeding ammonium lactate resulted in elevated rumen levels of acetic and propionic acids. These results agree with other work reporting the catabolism of lactic acid to acetic (Jayasuria and Hungate, 1959; Bruno and Moore, 1962) and lactic propionic acid (Elsden, 1954; Phillipson, 1952; Waldo and Schultz, 1956; Montgomery §E_gl., 1963). The rumen VFA data indicates the acid portion of ammonium salts is utilized and contributes to the energy available to the rumen microorganisms and the animal _143 However, it is not possible, at this time, to assess their mode of action or whether they are responsible for the greater utilization of ammonium salts than urea. Studies correlating rumen and rumen vein levels of the various VFA and ammonia with protein passing into the abomasum should be useful in determining their effect on ammonia utilization. SUMMARY The feeding value, metabolism and utilization of supplemented organic acids or ammonium salts of organic acids were studied in four feeding trials and three metabolic studies. The effect of high versus moderate levels of concentrate on the utilization of ammonium salts was also studied. Extensive research with urea has established its advantages and limitations, but there is only limited information available concerning other NPN sources. In zitrg studies have consistently demonstrated that ammonium salts of organic acids are equal or superior to urea as nitrogen supplements. Cattle fed ammonium salts have outperformed urea supplemented animals in the two trials that have been published (Varner et_al., 1968 and Varner and Woods, 1969). In Experiment I, ammonium acetate, ammonium lactate, soybean meal and urea were compared in a growth study on a ration composed of 75 per cent concentrates and 25 per cent corn silage. Cattle supplemented with ammonium acetate gained 5.4 per cent faster than cattle receiving soybean meal and 12 per cent faster than urea fed steers. The difference in average daily gain was. 144 145 significant (P < .05) between the ammonium acetate and urea fed steers. Ammonium lactate fed steers gained significantly (P < .05) faster than soybean meal (13.5 per cent) and the urea (20 per cent) fed groups. Cattle fed ammonium acetate consumed 3.1 per cent and 6.0 per cent more DM and showed 2.3 per cent and 6.1 per cent higher feed efficiency compared to the soybean meal and urea fed steers, respectively. Dry matter consumption was increased 9.1 per cent and 12.2 per cent for the ammonium lactate steers when compared to the soybean meal and urea fed steers, respectively. Similarly, feed efficiency was increased 3.5 per cent and 7.2 per cent. Although the difference was not significant, ammonium lactate fed steers were more efficient and had higher DM consumption and gains than ammonium acetate fed steers. Differences in carcass desirability were small and, for the most part, non-significant. In Experiment II, ammonium salts of formic, acetic, prOpionic lactic and butyric acids were compared to soy- bean meal, urea and a supplement that derived one-half of its protein equivalent from urea and one-half from corn steep water (U-CSW). The nitrogen supplements were added to either a ration of 40 per cent concentrates and 60 per cent corn silage or a ration of 80 per cent concentrates and 20 per cent corn silage. None of the differences in 146 gains were significant. However, the urea fed cattle had the lowest average daily gain, feed efficiency and DM consumption. The average daily gain of the groups fed the various ammonium salts was practically identical to the daily gain of the soybean meal and U-CSW supplemented groups when averaged over both concentrate levels (Table 18). However, the performance of all the NPN supplemented diets except ammonium acetate increased with a higher level of concentrate in the ration. The ammonium acetate fed cattle on the high concentrate ration gained 3.8 per cent less than those on the lower concentrate diet. The in- creased gains on the high concentrate rations compared to the rations low in concentrate were 2.9 per cent, 5.4 per cent, 4.2 per cent, 10.0 per cent, 2.6 per cent and 4.4 per cent for the U-CSW, urea, ammonium formate, ammonium propionate, ammonium lactate and ammonium butyrate fed groups, respectively. On the high concentrate ration, all of the NPN fed steers except urea and ammonium acetate outperformed the soybean meal fed steers. In contrast, the soybean meal fed steers outgained all other groups on the low level of concentrate. In addition, all of the NPN fed groups were more efficient than the soybean meal fed steers on the 80 per cent level of concentrate but less efficient than the soybean meal group on the low level concentrate. 147 The decreasing order of value of the nitrogen supplements, under the conditions of this experiment, was ammonium propionate, soybean meal, ammonium butyrate, ammonium lactate, ammonium formate, U-CSW, ammonium acetate and urea. The urea fed steers had the lowest carcass grade . '.. ' F3. and were the trimest in all fat measurements as well as in all carcass measurements that are correlated with the amount of carcass fat. The differences in all other carcass traits among the various nitrogen supplements, were small and non-significant. The average daily gain of cattle on high concen- trate diets was 2.9 per cent higher and the feed required per unit of gain was 7 per cent lower than on low concen- trate diets. However, the lower concentrate ration resulted in more beef production per hectare, a higher gross return per hectare and a lower cost of gain. Cattle -fed the high concentrate diet had a significantly higher carcass grade (P < .05) and dressing per cent (P < .01). In addition they were significantly (P < .01) fatter and had carcasses with significantly (P < .01) lower yields of boneless, trimmed retail cuts than those fed the low concentrate diet. Experiment III was designed to study the effect of the addition of lactic or acetic acid in equalmolar concentrations as contained in the ammonium salts fed in 148 Experiment I. The acid additions were compared in both urea and soybean meal supplemented rations. Adding acetic acid decreased feed efficiency 3.7 per cent but adding lactic acid increased feed efficiency 2.6 per cent. The addition of lactic tended to reduce the amount of carcass fat. Urea fed cattle had a decrease of 4.3 per cent, 2.6 per cent and 1.9 per cent in gain, consumption and feed efficiency, respectively, when compared to soybean meal fed steers. Fat thickness of the urea supplemented steers was significnatly (P < .05) lower than for steers fed soybean meal. In Experiment IV, the addition of acetic or lactic acids in increasing increments to all silage rations was studied. The addition of acetic acid to the ration de- pressed gain and feed efficiency for all levels of acetic acid addition compared to the control.. However, none of these differences were significant (P < .05). Average daily gain was not affected by adding lactic acid to the ration. There was also no difference in feed efficiency or consumption for the various levels of added lactic acid. The addition of lactic acid did result in a de- creased consumption of the basal ration (silage and protein), but this decrease was offset by the lactic acid intake. Experiment V involved stomach pumping and bleeding cattle that had previously been on Experiments I and III. . 1.15-1, 8W ‘1'; _. 149 The rumen ammonia concentrations were highest at T2 5 (2.5 hours post feeding) for urea, ammonium acetate and ammonium lactate. The ammonium lactate cattle also had 5 and T10. The blood urea levels were higher at all determinations for higher (P < .05) rumen ammonia levels at T the cattle fed ammonium salts. At T5, blood urea levels i were significantly greater for cattle fed ammonium lactate (P < .01) or ammonium acetate (P < .05) than for cattle fed urea or soybean meal. Rumen acetate concentration was higher at all 3 determinations for cattle fed ammonium lactate than for cattle receiving ammonium acetate or urea. Rumen acetate levels were higher for steers fed ammonium salts at T2 than for those fed soybean meal or urea. Rumen propionate levels tended to be higher for the steers fed soybean meal and ammonium lactate at all sampling times and were significantly higher (P < .01) at T2.5 for the ammonium lactate fed cattle than for any other group. Rumen butyrate was not detected in rumen fluid samples of the ammonium lactate fed steers. Rumen butyrate levels were signifi- cantly higher (P < .05) for cattle fed soybean meal than for those fed ammonium acetate or urea. When lactic acid was added to the ration, the rumen ammonia level as well as the blood urea level, was higher at all sampling times. Cattle receiving acetic acid in the diet had the highest rumen acetate level at 150 T2.5' Lactic acid fed steers had the highest rumen ace- tate (except T2.5) as well as the highest rumen propionate levels at all sampling times. Rumen butyrate levels were highest for the control groups and was not detectable when urea was fed in combination with lactic acid. In Experiment VI, nitrogen balance and metabolic studies were conducted with fistulated steers fed the same rations as Experiment I. In contrast to Experiment V, the rumen ammonia and blood urea level of the urea fed steers was higher at all sampling times except T10 than for the soybean meal or ammonium salt fed cattle. As in Experiment V, supplementation with ammonium lactate increased the levels of rumen acetate and propionate. Rumen butyrate levels were higher for steers fed ammonium salts than for steers fed urea or soybean meal. The ammonium acetate fed cattle had the lowest DM consumption. The ammonium lactate fed steers had the highest DM digestibility (72.6 per cent). They were followed by ammonium acetate (69.7 per cent), soybean meal (67.9 per cent and urea supplemented rations (64.6 per cent). The steers fed ammonium salts had a greater nitrogen intake but did not lose any more nitrogen in the feces or urine than the soybean meal and urea fed steers. This resulted in an increased quantity of nitrogen digested and a significantly (P < .01) higher nitrogen balance for the steers fed ammonium salts. It? 151 Experiment VII was a nitrogen balance and meta- bolic study of the nitrogen supplements in Experiment II on the 40 per cent concentrate — 60 per cent corn silage ration. Rumen ammonia concentrations were maximized at T2 and were highest for ammonium lactate and ammonium acetate fed steers. Steers fed natural protein (soybean meal) had the highest rumen ammonia values at T8 and T10. There was no significant difference among the various lnitrogen sources tested for blood urea levels. Rumen acetate concentrations were higher for ammonium acetate fed steers at T but lower than soybean 2 meal, ammonium lactate, U-CSW, ammonium formate and ammonium butyrate at T4. Supplementing the diets with soybean meal or ammonium lactate resulted in higher rumen prOpionate levels. Rumen propionate concentration was higher for ammonium propionate fed steers at T2 than for steers receiving other supplements except ammonium lactate. Rumen butyrate was higher at T2, T4 and T6 for the ammonium butyrate fed steers. Ammonium lactate and soybean meal fed steers also had elevated rumen butyrate levels at T4. Differences in rumen pH were small and insignificant except that rumen pH was significantly lower at T4 for soybean meal fed steers. None of the differences in DM intake or digestr ibility were significant. Soybean meal fed steers had the lowest dry matter digested. There were no significant 152 differences in any of the nitrogen parameters studied. All nitrogen sources except soybean meal promoted positive nitrogen balances. Daily nitrogen intake was highest for the U-CSW fed steers, lowest for the soybean meal steers and intermediate for cattle fed urea or ammonium salts. As in Experiment VI, urine and fecal nitrogen losses were relatively constant and the decreased nitrogen intake on the soy ration was probably the major cause of the negative balance. The results of these experiments indicate that ammonium salts of organic acids are superior to urea and in many cases equal or superior to soybean meal in pro- moting efficient beef gains and positive nitrogen balance. Ammonium acetate was the poorest ammonium salt tested. The acid portion of ammonium salts is an efficient energy source and does not, with the exception of ammonium acetate, effect DM consumption. BIBLIOGRAPHY ‘ 3:27 BIBLIOGRAPHY Acord, C. R., G. E. Mitchell, Jr., C. 0. Little and Mr. Kerr. 1966. Nitrogen sources for starch di- gestion by rumen microorganisms. J. Dairy Sci. 49:1519. Allen, C. K. 1972. The economic feasibility of supple- menting brood cows and yearling steers with protein, minerals and vitamins while grazing frosted winter pastures. M.S. Thesis Mich. State Univ. Allison, M. 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